use of nutritional supplementation to improve responses · on average, 50% of the flock experiences...
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Use of nutritional supplementation to improve responses
to the ‘ram effect’ in the Merino ewe
Phillip Clemens Khaiseb
Supervisors:
Professor Graeme B. Martin (University of Western Australia, Perth)
Dr Penny A. R. Hawken (University of Western Australia, Perth)
A thesis submitted to fulfil the requirements for the degree of Master of Agricultural
Science
School of Agriculture and Environment
University of Western Australia, Perth, Australia
December 2016
Table of Contents
Declaration ..................................................................................................................................................................... i
Acknowledgements .................................................................................................................................................. ii
Summary ........................................................................................................................................................................ iii
Chapter 1 ........................................................................................................................................................................ 1
1.1 General Introduction ....................................................................................................................................... 1
Chapter 2 ........................................................................................................................................................................ 4
2.1 Review of the literature .................................................................................................................................. 4
2.2 The Reproductive System.............................................................................................................................. 4
2.2.1 The hypothalamo-hypophyseal axis ......................................................................................................... 4
Figure 1: Schema of the hypothalamic-pituitary axis in sheep. .................................................. 5
2.3 Folliculogenesis .................................................................................................................................................. 5
Figure 2: The structure of an ovary. ............................................................................................................. 6
Figure 3: A model for folliculogenesis in the ewe. ............................................................................... 7
2.4 Primordial follicles ............................................................................................................................................ 8
2.5 Committed follicles ........................................................................................................................................... 8
2.6 Gonadotrophin-responsive follicles ............................................................................................................ 8
2.7 Gonadotrophin-dependent follicles ............................................................................................................ 8
2.8 Ovulatory follicle ............................................................................................................................................... 9
2.9 Steroidogenesis ............................................................................................................................................... 10
Figure 4: A pictorial illustration of ovarian steroidogenesis. ...................................................... 10
2.10 Feedback Mechanisms .................................................................................................................................. 11
2.11 The negative feedback .................................................................................................................................. 11
2.12 Positive feedback ............................................................................................................................................ 12
Figure 5: Negative and positive feedback mechanisms. ................................................................ 12
2.13 The Oestrous Cycle ........................................................................................................................................ 13
Figure 6: Oestrous cycle of the ewe. .......................................................................................................... 13
2.14 “Male effect” for controlled breeding....................................................................................................... 14
2.15 Isolation of ewes from males ..................................................................................................................... 15
2.16 Ewe response to the “male effect” ........................................................................................................... 15
2.17 The short cycle ................................................................................................................................................ 17
2.18 Nutrition ............................................................................................................................................................. 17
2.18.1 Types of nutrition ........................................................................................................................................... 18
2.18.1.1 Acute effect ................................................................................................................................................ 18
Figure 8: Acute effect of feeding. ............................................................................................................... 19
2.18.1.2 Static effect ................................................................................................................................................ 19
2.18.2 Acute versus Static Effect ........................................................................................................................... 20
2.18.2.1 Dynamic effect .......................................................................................................................................... 21
2.19 Nutrition to increase ovulation rate ......................................................................................................... 21
2.20 Nutrition to improve response to the male effect .............................................................................. 22
2.21 Summary ........................................................................................................................................................... 22
Chapter 3 ...................................................................................................................................................................... 24
3.1 General Methods and Materials ................................................................................................................. 24
3.1.1 Area of study ................................................................................................................................................... 24
3.2 Experiment 1 .................................................................................................................................................... 24
3.3 Experiment 2 .................................................................................................................................................... 25
Chapter 4 ...................................................................................................................................................................... 27
4.1 Introduction ...................................................................................................................................................... 27
4.2 Materials and Methods .................................................................................................................................. 29
4.3 Part 1 ................................................................................................................................................................... 30
4.4 Part 2 ................................................................................................................................................................... 31
4.5 Results ................................................................................................................................................................ 31
Table 1: Reproductive variables: ................................................................................................................. 33
Figure 11: Oestrus distribution curves in ewes. ................................................................................. 33
Table 2: Singles, twins and overall pregnancy rates: ...................................................................... 34
4.6 Discussion .......................................................................................................................................................... 35
Chapter 5 ...................................................................................................................................................................... 38
5.1 Introduction ...................................................................................................................................................... 38
5.2 Methods and Materials .................................................................................................................................. 39
5.3 Experimental design ...................................................................................................................................... 40
Figure 12: Schematic experimental design. .......................................................................................... 40
5.4 Results ................................................................................................................................................................ 41
Figure 13: Average live weight of ewes................................................................................................... 41
Table 3: Reproductive variables: .................................................................................................................. 42
Figure 14: Oestrus distribution of anovular ewes induced at the ‘male effect’. .............. 43
5.5 Discussion .......................................................................................................................................................... 44
Chapter 6 ...................................................................................................................................................................... 47
6.1 General Discussion ......................................................................................................................................... 47
References ................................................................................................................................................................... 50
ii
Acknowledgements
I wish to take this opportunity to sincerely thank Graeme Martin for his enormous
patience with me through unwavering guidance and wisdom he passed on to me. I have
learned a great deal from him for his art in writing, especially communication in
science. It requires absolutely different skills altogether to communicate scientifically,
but in a simple manner to ensure that the reader understands everything – and it is
something which I shall continue to learn. Penny Hawken has been a silent contributor
to my learning process during my time at UWA, but she has been very effective in
sharing her knowledge with me. I valued her teaching which was in an unstressed
environment and I fully came to grasp with the reproductive physiology in mammals
during my first year. Thank you to both Graeme and Penny and memories will remain
with me forever. Importantly, I would like to thank the AUSAID Scholarship for having
granted me this once in a life time opportunity to upgrade myself academically and
having spent 24 months at UWA, which I learned immensely both academically and
socially. Finally, to my beloved wife, Fealofani Khaiseb, my two daughters, Leilani A.
Khaiseb and Anisa T. Khaiseb. You allowed me to endeavour this project and you were
also very understanding not to spend long periods with you for me to achieve my
objectives. I am indebted to you for all the support you afforded me in whatever shape
and forms they came. Fani, you also pushed me to get this thesis behind me, thanks a lot
for that.
iii
Summary
The pressure from world’s societies to develop technologies in animal production systems
has prompted researchers around the world to focus on three things. We use an approach that
is hormone-free, environmentally friendly as well as taking care of animal welfare. This
approach has led to the development of ‘clean, green and ethical’ concept in Australia. The
important tool to induce ovulation in anovular ewes which were previously isolated from ram
induces a physiological response, a phenomenon coined the ‘ram effect’. This effect is
sufficient to override the seasonal suppression of the hypothalamic-pituitary axis to induce in
the ewes.
The objective in the first experiment was to develop a regime for short-term nutritional
supplementation before and after ram induce oestrus cycle at the onset of the natural breeding
season to improve reproductive performance of ewes. Nutritional supplementation was not
possible to test as ovulation was observed in all ewes with only 6% of ewes which did not
ovulate in the controls. However, there was an increase in the ovulation rate in the group
which received nutritional supplementation during the ram induced oestrous cycle (luteal
phase). The synergistic effects of the ‘ram effect’ and nutritional supplementation did not
reduce the proportion of ewes experiencing a short cycle. The experiment showed that
nutritional supplementation seems to have a greater effect on follicle stimulation during the
oestrous cycle.
The second experiment was conducted to investigate the effect of short-term nutritional
supplementation on the response of anovular ewes during the non-breeding season following
the ‘ram effect’. Similar to the first experiment, ovulation was poor in the treatment group
while the controls showed low ovulation which confirms that nutritional supplementation
does not affect ovulation. The ovulation rate was increased in the treatment group at the first
ovulation, which was evident on Day 10, but it subsequently dropped to the similar level of
the controls once the nutritional supplementation was discontinued. Therefore, the results of
the first and second experiments confirm that nutritional supplementation is critical just prior
to the preovulatory period which suggest that the length rather than the timing of the
nutritional supplementation is important. The luteal failure is independent of the nutritional
stimulus because there is consistency in the increased ovulation rate at the first ovulation and
the expected stimulation of folliculogenesis by the lupin supplements.
iv
In conclusion, we have demonstrated through the repeatability of two separate experiments at
the onset of the breeding season and during the non-breeding season that nutritional
supplementation does not have an effect on the number of ewes ovulating, but increases
ovulation rate. Such supplementation should be sustained until the preovulatory stage at the
ram induced oestrous cycle.
1
CHAPTER 1
1.1 General Introduction
Namibia is situated in the south-west of the African continent bordering the Atlantic Ocean
on the west. Rainfall has a wide regional variation, from less than 20 mm in the western
Namib and coastal zones to more than 700 mm at the eastern end of the Caprivi strip (Sweet,
1998). The communal farmers occupy 41% of the total land area, in regions that receive less
rain relative to that occupied by commercial farmers, who occupy ‘prime land’ about 44% of
total land area. The production systems in the communal areas are based on pastoralism and
agro-pastoralism, and the majority of households are subsistence-based and labour intensive
with limited use of technology and external inputs. It is important to improve small ruminant
production in these subsistence systems so these farmers can access high price markets.
The main problems that subsistence farmers encounter are: (i) poor or no management
strategies for feeding leading to livestock in poor condition at mating; (ii) no subdivision of
land into paddocks to ensure that males are kept separate from females during the non-
breeding season; (iii) also, as a result of the lack of subdivision, there is overgrazing, with
effects exacerbated by erratic rainfall; and, (iv) low income that prevents the use of expensive
technologies. Therefore, it is logical for sheep and goat farmers in semi-arid and arid
conditions to adopt low cost management strategies that give them greater reproductive
output, yet also allow flexibility so mating and lambing can be timed to coincide with
abundant food. In current Namibian conditions, farmers simply rely on the natural seasonality
of their animals to dictate the onset and succession of the breeding season.
The hierarchy of factors controlling the onset of reproductive activity varies greatly around
the world. Photoperiod is the primary driver of reproductive activity and in high latitudes; it
dominates completely, leading to strict patterns of seasonal breeding (Adams, N. R.;
Atkinson, S.; Martin G. B.; Briegel, J. R.; Bouklig, R.; Sanders, 1993). In regions closer to
the Equator, the power of photoperiod fades so rainfall and associated abundance of food
dictate the onset of the breeding season (Martin et al., 2004b). Breeds of sheep and goats
native to lower latitudes, such as Merinos, are very opportunistic and the onset of
reproductive activity is equally affected by changes in food availability and reduced day
length (Chemineau et al., 1988). Effective coordination of reproductive opportunistic
breeding patterns will still be hampered by year-to-year variations in the timing of the rainy
periods, but they offer the best option for farmers in regions such as Namibia.
2
It is important for late pregnancy and lactation to coincide with most of the rainy season. In
Namibia, this generally means that the breeding season should commence in spring instead of
autumn. For genotypes that are strongly controlled by photoperiod, the peak in annual food
supply will be “out of phase” (Martin et al., 1994) . Although mating out of the breeding
season can lead to lower lambing percentage, better lamb survival rates will be achieved
(Parsons and Hunter, 1965). Mating in spring is feasible because social-sexual stimuli are
also able to induce the reproductive activity by over-riding both circannual rhythm and the
inhibitory effects of photoperiod (Martin et al., 2002; Martin et al., 1999). Controlled
breeding using the ‘male effect’ could therefore play an important role in strategies to
improve reproductive performance where “out of phase” breeding is advantageous.
The ‘male effect’ is predominantly of pheromonal stimulus that increases LH pulse frequency
and stimulates the growth of ovarian follicles, leading to ovulation (Atkinson and
Williamson, 1985). However, it is not an effective tool on its own because the proportion of
ewes ovulating, ovulation rate and proportion of ewes experiencing a short cycle are variable
from season to season. On average, 50% of the flock experiences a normal cycle and the
other 50% a short cycle. The short cycle is the result of poorly developed corpus luteum
which only lasts for 6 to 9 days at second ovulation (Martin, 1979; Oldham and Martin,
1979). It is important to improve or prevent the occurrence of the short cycle because it will
allow farmers to have better control over timing of the breeding season, planning of
pregnancy and subsequently the lambing season to coincide with the food availability.
The general theory is that the problem is at the ovary level where the preovulatory follicles do
not develop fully to produce a viable corpus luteum. The sudden introduction of rams causing
acute LH pulse frequency and secretion within 10 minutes, provides insufficient time for the
transformation of the gonadotrophin-dependent follicle into an ovulatory follicle. The
concept “window of opportunity” was developed which is understood to be the period of time
when the sensitivity of the gonadotrophin-dependent follicle to FSH stimulus is adequate so
that it will resist atresia and develop into an ovulatory follicle (Scaramuzzi et al., 1993).
The response to the “ram effect” can be improved with nutritional supplementation (Stewart
and Oldham, 1986; Oldham and Lindsay, 1984; Knight et al., 1983; Knight et al., 1979;
Lightfoot and Marshall, 1976; Lightfoot et al., 1976). Nutritional supplementation stimulates
follicle growth and the follicles will be afforded that “window of opportunity” to grow further
even with little amounts of FSH concentrations. The overall effect of short-term lupin
3
supplementation is the immediate increase of insulin concentrations after feeding that leads to
increased supply of glucose to the follicles, mediating nutritionally-induced stimuli in
ovulation rate (Downing et al., 1995). Therefore, nutritional supplementation before the “ram
effect” may play an important role in preventing the short cycle, hence, synchrony of the
majority of the ewes within a three-day period.
In 1995, the Farming Systems, Research and Extension (FSRE) programme was implemented
by the Ministry of Agriculture, Water and Forestry (MAWF) to improve the efficiency and
productivity of sheep and goat farming in Namibia. The FSRE aims to make research
outcomes more accessible to farmers, since most research into reproductive strategies is done
under controlled conditions and not necessarily directly applicable to farming systems.
Namibian farmers are in an ideal position to benefit from the increasing demand for “clean,
green and ethical” (CGE) agriculture (Martin et al., 2004a) since their management strategies
are extensive and rarely use hormones or chemicals.
The proposed research project will investigate how two CGE management tools, the ‘male
effect’ and focussed feeding can be used to influence the reproductive performance of small
ruminants. I am optimistic that this information will help me to develop strategies for
controlling reproduction in sheep that are both directly applicable and easily accessible to
communal farmers in Namibia. The general hypothesis is that nutritional supplement will
improve the response to the ‘male effect’ and the subsequent reproductive performance of
Merino ewes.
4
CHAPTER 2
2.1 Review of the literature
In the hypothalamic-pituitary axis, there is a convergence of various external and internal
inputs that influence outputs that control many essential bodily functions, including
reproduction. For reproduction in the ewe, for example, the external and internal inputs are
processed and the outcome determines the pattern of secretion of the neurohormone,
gonadotrophin-releasing hormone (GnRH) which, in turn, regulates the secretion of the
gonadotrophins from the pituitary gland. The gonadotrophins stimulate the ovaries which
then send signals (primarily sex steroid hormones) back to the brain to exert feedback on
GnRH/gonadotrophin secretion and elicit sexual behaviour. This system, linking
hypothalamus, pituitary gland and ovaries, is responsible for the generally close temporal
relationship between ovarian function (ovulation) and sexual behaviour.
In addition to the primary sex steroids (oestrogens and progestagens), the internal factors
include metabolites and metabolic hormones that convey information to the reproductive
control centres about body reserves (including the demands of lactation) and stress responses.
Among the external factors are those that reflect season (night-length) and nutrition, to which
the reproductive centres generally respond by ensuring that lambing occurs at that time of the
year when the food supply is adequate for offspring survival.
This review of the literature discusses the physiological processes that are linked to the
control of reproduction in the female, with a focus on sheep, and explores the possibility of
using nutritional management to improve the reproductive performance.
2.2 The Reproductive System
2.2.1 The hypothalamo-hypophyseal axis
Reproductive activity is primarily controlled by the activity of the neurons in the brain that
produce and secrete gonadotrophin-releasing hormone (GnRH). In sheep, the GnRH cell
bodies are located in the hypothalamic-preoptic continuum (Caldani et al., 1988). The
synthesis of GnRH by these neurones is transported along the axons to the median eminence
where it is released, in pulses rather than as a continuous stream, into the hypophyseal portal
system that connects with the anterior pituitary gland (Fig. 1). The arrival of GnRH pituitary
tissue stimulates the synthesis and secretion of the gonadotrophins, luteinizing hormone (LH)
and follicle-stimulating hormone (FSH), two of the major regulators of gonadal function
5
(Lincoln, 1979) that are then transported to the target tissues, the ovaries, to stimulate
gonadal activity.
Gonadotrophins released in
peripheral blood
Gonadal
activity
LH
FSH
Pituitary Gland
Anterior
Pituitary
gland
Optic
Chiasma
GnRH
Neurons
Preoptic-hypothalamic
continuum
Median Eminence
Terminals
AHA
MBH
GnRH
Figure 1: Schema of the hypothalamic-pituitary axis in sheep.
The pulsatile release of GnRH stimulates the secretion of LH pulses (on a 1:1 basis), whereas
it elicits a more continuous stream of FSH secretion. In the brain, the important regions are
the preoptic area (POA), anterior hypothalamic area (AHA), the mediobasal hypothalamus
(MBH) and the median eminence. Adopted from Thiéry & Martin (1991) and Caldani et al.
(1988).
2.3 Folliculogenesis
Folliculogenesis, the process of follicle growth and development, is a complex series of
events leading to an increase in number of follicles through the differentiation of somatic and
germ cells within the ovary. This proliferation of follicles commences in late stages of foetal
development, and the subsequent maturation of small cohorts takes place continuously,
through puberty and throughout the adult life. The maturation phase takes 4-6 months in
cattle and sheep (Hunter et al., 2004b; Souza, Campbell & Baird, 1997), but just over three
months in pigs (Hunter et al., 2004a). The final stages of maturation are dynamic and rapid
and, in the sheep, culminate in the production of only 1-3 (usually) mature, ovulatory
(‘Graafian’) follicles during the follicular phase of the oestrous cycle (Fig. 2; Pineda, 2003a).
6
The whole process, from differentiation of large numbers of primordial follicles through to
the ovulation of a few at the end of each oestrous cycle, is driven by numerous signalling and
gene expression events (McNatty et al., 2006). The follicle tissues (theca and granulosa cells)
and the oocyte that each follicle carries, communicate continuously with each other though
molecules such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15
(BMP15), both secreted in the oocyte, sustain development and maturation of the follicle
(McNatty et al., 2006; McNatty et al., 2004).
The initiation of growth in a primordial follicle involves many multiplications of granulosa
cells which eventually form more than one layer around the oocyte (Fig. 2) and ultimately
create an environment in which an antrum forms, a space full of fluid (Webb & Campbell,
2006; McNatty et al., 2006). The antral follicles in sheep range between 200 and 400 μm in
diameter (Turnbull, Braden & Mattner, 1977). The antral fluid contains large quantities of
ovarian steroids (androgens, oestrogens, progestagens), electrolytes, growth factors and
cytokines (Pineda, 2003a). As a result of the increasing volume of the follicular fluid in the
antrum, the follicle begins to bulge from the surface of the ovary.
Figure 2: The structure of an ovary.
Clockwise changes during the oestrous cycle of the sheep, starting at the hilum as it recurs on
average every 16-17 days. Primordial follicles go through a complex maturation process that ends
with atresia for most of them, and ovulation of a mature oocyte for a few of them. As the preovulatory
follicle enlarges, it secretes large amounts of oestrogen and inhibin and these two hormones act
synergistically to suppress the development of other follicles through inhibitory feedback on FSH
secretion (this is ‘follicle selection’ and leads to ‘follicle dominance’). Following ovulation, the
remnants of the follicle are transformed into a corpus luteum (luteinization). Adapted from Porterfield
(2001a).
7
After ovulation, when the ovum is released for potential fertilisation by the sperm, the corpus
luteum forms and, if there is no pregnancy, regresses to allow a new cycle to begin.
The process of follicular development is controlled by a highly co-ordinated endocrine and
paracrine signals that ultimately determine whether a follicle ovulates and also whether more
than one follicle achieves this outcome (ovulation rate). The endocrine signals are between
the pituitary gland and the ovary, whereas the paracrine signals are among the follicular cells,
viz. the oocyte, cumulus, granulosa and thecal cells (Juengel & McNatty, 2005; Shimasaki et
al., 2004; Matzuk et al., 2002). The process of follicular development is continuous but is
nowadays divided into functional (rather than morphological) stages: primordial, committed,
gonadotrophin-responsive, gonadotrophin-dependent and ovulatory (Fig. 3). These stages
also involve changes in capacity for steroidogenesis, an integral part of the ovarian cycle.
Figure 3: A model for folliculogenesis in the ewe.
Cascade of development during which follicles emerge from a pool of primordial follicles to
enter a process of growth and development that is continuous and ends in either atresia or
ovulation. In most mammals, this process is approximately linear to the gonadotrophin-
responsive stage and, especially in ruminants, becomes wave-like in the gonadotrophin-
dependent stage. Adapted from Scaramuzzi et al. (2011).
Primordial follicles (Type 1 or 1a): Largest population of follicles present, oocyte and granulosa cells have
receptors for some growth factors, but not LH or FSH
Committed follicles (Types 2-3; primary and small preantral): FSH and LH not essential; continuous growth
controlled by the oocyte; low rate of atresia, development
of zona pellucida
Gonadotrophin-responsive follicles (Types 4 and 5; large preantral and small-medium antral): Development
of LH-responsive theca interna and FSH-responsive
granulosa cells; oocyte-mediated differentiation of a
cumulus phenotype; increasing atresia and progressive
increase in requirement for gonadotrophins
Ovulatory follicle(s): have LH receptors on granulosa cells; can survive with low FSH
concentrations
Ovulation: Initiated by LH surge and
occurs some 72 h later
Atresia
Antrum forms
Gonadotrophin-dependent follicles (Type 5+; medium-large antral): Become atretic if FSH concentrations are
low before granulosa cells develop LH receptors; high
rate of atresia associated with follicle waves
8
2.4 Primordial follicles
Folliculogenesis begins with the development in the foetus of a pool of quiescent primordial
follicles that are about 0.03 mm in diameter (Scaramuzzi et al., 2011). With little selection
taking place, these follicles are primarily gonadotrophin-independent (Lopez-Sebastian et al.,
1993; Cahill & Mauléon, 1980; Dufour, Cahill & Mauléon, 1979; Turnbull et al., 1977;
Hutchinson & Robertson, 1966). Young ewes have between 40,000 to 300,000 primordial
follicles.
2.5 Committed follicles
In an apparently ordered sequence, gradually and regularly, primordial follicles leave the
resting pool and start to grow under the control of stimulatory and inhibitory feedback
mechanisms between the ovaries and the hypothalamo-hypophyseal axis (Cahill, Mariana &
Mauléon, 1979). On entering this phase, they are ‘committed’ to full-scale development that
will, ultimately, end in atresia for all except the few that ovulate, although very few die at this
early stage. The process seems to have evolved to ensure that high numbers of follicles which
enter the gonadotrophin-responsive stage, increasing the likelihood of recruitment for
ovulation.
2.6 Gonadotrophin-responsive follicles
After becoming committed, the follicles become gonadotrophin-responsive and develop
receptors for LH on the theca cells and for FSH on the granulosa cells (Scaramuzzi et al.,
1993, 2011). There is some doubt about whether the gonadotrophins are essential for
development beyond the committed stage, but the follicles are classed as gonadotrophin-
responsive because they can respond to gonadotrophins by synthesising cyclic adenosine
monophosphate (cAMP) and steroids (McNatty et al., 1992). Furthermore, the granulosa cells
synthesise and secrete proteoglycans in response to FSH, suggesting that they are perhaps
involved in the formation of the antrum by preventing early luteinization in the later stage of
the antral follicle development (Findlay et al., 1993; Baird, 1984; Ax & Ryan, 1979). About
25 follicles with diameters of 1.0-2.5 mm emerge from this phase, but most are apparently
too immature to be gonadotrophin-dependent and thus ovulate in the absence of exogenous
gonadotrophins (Henderson et al., 1988; Tsonis et al., 1984).
2.7 Gonadotrophin-dependent follicles
In this phase, the follicles develop an antrum and become tertiary and Graafian follicles in a
process that leads to wave-like patterns of follicle growth during the cycle primarily under
9
gonadotrophic control (Bartlewski et al., 1998; Ginther, Kot & Wiltbank, 1995; Ginther &
Kot, 1994; Ravindra et al. 1994; Noel, Bister & Paquay, 1993). The waves are due to
interactions between FSH, inhibin and oestradiol, and are associated with the phenomenon of
follicle ‘dominance’ (Viñoles et al., 2010; Scaramuzzi et al., 2011). For the follicles to grow
larger than 2.5 mm in diameter, they need FSH and LH and, because the largest follicle(s)
produce the most oestradiol and inhibin A, they inhibit FSH secretion and drive other
gonadotrophin-dependent follicles into atresia (Scaramuzzi et al., 1993; Gonzalez-Añover et
al., 2007). The dominant follicles have very high concentrations of oestradiol in their
follicular fluid so they are often classified as ‘oestrogenic’ (Fortune, Rivera & Yang, 2004;
Scaramuzzi et al., 1993; McNeilly, Jonassen & Fraser, 1986).
This, however, is a critical phase for the dominant follicles too – they are also totally
dependent on gonadotrophic control to avoid atresia so they overcome the decreasing
concentration of FSH by shifting their dependency to LH; they develop LH receptors on the
granulosa cells and thus no longer need FSH to produce aromatase and convert androgen to
oestrogen (Campbell, Scaramuzzi & Webb, 1995). Because of their vulnerability to atresia,
and thus the process of follicle selection, the number of gonadotrophin-responsive and
ovulatory follicles declines markedly and only 1-8 follicles remain (Scaramuzzi et al., 1993).
2.8 Ovulatory follicle
In the final stages of the follicular phase of the oestrous cycle, FSH concentrations are very
low but the actual values might still be critical for final maturation of the granulosa cells
(Henderson et al., 1988). Variations in the FSH concentration at this time are probably due to
variability within and between animals (Scaramuzzi et al., 1993). The granulosa cells in
ovulatory follicles present with large number of LH receptors and they become highly
responsive to both LH and FSH (Webb & England, 1982). The aromatase activity per
granulosa cell reaches a maximum, leading to massive increases in the production and
secretion of oestradiol and inhibin, finally suppressing FSH concentrations below the critical
threshold required by the gonadotrophin-dependent follicles, all of which undergo atresia
(Scaramuzzi et al., 1993). In rats, the oestradiol also increases the responsiveness of the
ovulatory follicle to gonadotrophins by enhancing the ability of FSH to induce LH and FSH
receptors (Richards, 1980). Furthermore, in humans and rats, local peptides like inhibin
enhance the ability of LH to stimulate androgen production by thecal cells (Hillier et al.,
1991; Hsueh et al., 1987).
10
At this stage, only a few follicles remain and enter the ovulatory process. This number
controls the ovulation rate of the animal – for a Merino ewe, usually, only one or two ova are
shed at ovulation.
2.9 Steroidogenesis
Implicit in folliculogenesis is the synthesis of sex steroids by the theca and granulosa cells
(Fig. 4), a process that is best explained by the two-cell theory in which the theca and
granulosa cells complement each other (Falck, 1959). Importantly, the granulosa cells cannot
produce the androgens as they lack the enzyme 17α-hydroxylase. Therefore, the theca interna
cells produce androgen as the precursor for oestrogen production by aromatase in the
granulosa cells (Porterfield, 2001a). The thecal cells have LH receptors (LH-R) linked to
17α-hydroxylase activity throughout all stages of antral follicular development (Porterfield,
2001a; Hillier, 1985) and thus androgenic steroids are synthesised during active periods of
antral growth (McNatty, 1982; Moor, 1977). The granulosa cells, on the other hand, are the
predominant site of oestradiol-17β production and are the only cells that have FSH receptor
(FSH-R) through which FSH stimulates the production of inhibin and the production of
oestrogen by aromatization of androgens that have diffused in from the theca cells (Pineda,
2003a; Hillier, 1985; Porterfield, 2001a).
Theca
cells
Antrum
Oocyte
Cholesterol
Pregnenolone
Progesterone
17-hydroxyprogesterone
Testosterone
Androstenedione
Testosterone
Oestradiol
LH-R
LH
FSH
Granulosa
cells
Aromatase
FSH-R
Androstenedione
DiffusionLH-R
Figure 4: A pictorial illustration of ovarian steroidogenesis.
From the precursors (cholesterol pregnenolone, progesterone) theca cells produce
androgens that then diffuse into the granulosa cells where they are converted to oestrogens
by aromatase. In antral follicles, the granulosa cells develop LH receptors so do not need to
rely on FSH for the production of aromatase.
11
2.10 Feedback Mechanisms
Ovarian function is dependent on the actions of pituitary LH and FSH to stimulate follicular
growth, and the secretion of LH and FSH is, in turn, controlled by inhibitory and stimulatory
feedback through a range of interactions that orchestrate the stages of the reproductive cycle
(Pineda, 2003b).
2.11 The negative feedback
The negative feedback occurs when the concentration levels of progesterone, oestradiol and
inhibin are sufficiently high to suppress the response to the hypothalamic-pituitary axis.
Progesterone synthesised and secreted from the corpus luteum synergises with oestradiol-17
to inhibit hypothalamus for the stimulation of GnRH.
It has been demonstrated that either oestradiol or progesterone alone cannot inhibit the LH
release and that they require a collective effort (Martin & Thomas, 1990). The ovarian
oestrogens and progestagens as well as other locally produced ovarian peptide factors
(inhibin A and follistatin) feed back to the hypothalamus and pituitary to regulate the
secretion of the gonadotrophins (Fig. 5) (Findlay et al., 1993). Particularly, inhibin which is a
dimeric polypeptide factor, acting directly on the anterior pituitary gland and oestradiol-17
which acts on both the hypothalamus and the anterior pituitary gland inhibit the secretion and
release of LH and FSH by adenohypophysial cells (Pineda, 2003a).
The feedback in the female is complicated by the combinations that can arise because there
are two endocrine organs in the ovary that produce different hormones with different targets
in the hypothalamo-pituitary axis.
Negative Feedback Loop 1 Gonadotrophin-releasing hormone pulse frequency is inhibited
by oestrogen (from the follicles) and progesterone (from the
corpora lutea) acting synergistically on the hypothalamus;
Negative Feedback Loop 2 Follicle-stimulating hormone secretion is inhibited by oestrogen
and inhibin acting synergistically on the anterior pituitary
gland.
Between them, these two negative feedback loops can explain nearly all the events of the
female ovulatory cycle. For instance, the transition from the luteal phase (when progesterone
is present) to the follicular phase (when progesterone has disappeared) is explained by loss of
12
Feedback Loop 1. This allows GnRH pulse frequency to ‘escape’, stimulate the secretion of
LH and FSH, and drive follicular growth and maturation. As the follicles grow and mature,
they produce lots of oestrogen, but oestrogen is a very weak agent of negative feedback, so
GnRH pulse frequency is not affected.
Feedback Loop 2 is most important during follicle selection because the main source of
oestradiol and inhibin, the dominant follicle(s), drive FSH secretion down, depriving other
follicles of this essential hormone and thus causing atresia.
As oestrogen builds up during the follicular phase it triggers a ‘switch’ in the hypothalamus
and pituitary gland and a large surge of GnRH, LH and FSH is produced which drives the
last stage of follicle development and leads any fully mature follicles to burst and release
their ova. The high concentration of oestrogen at this time also induces oestrous behaviour.
2.12 Positive feedback
The secretion of FSH stimulates growth and differentiation of ovarian follicles during the
preantral and early antral stages. Unlike LH, however, FSH release is not in pulses but it
occurs in a constant manner (Martin, Rodger & Blache, 2004).
For the release of LH, each GnRH pulse results in an equivalent pulse of LH and each pulse
of LH, in turn, stimulates the follicles to secrete a pulse of oestrogen (Martin, 1984). The
effect of LH is two-fold: i) it acts on theca interna cells and luteal cells; and, ii) late in the
follicular phase, it acts on granulosa cells to sustain further growth of the large antral follicles
(oestrogenic) (Porterfield, 2001a; Baird et al., 1991; Baird, 1984).
Figure 5: Negative and positive feedback mechanisms.
A schematic illustration of the regulation of ovarian function via negative and positive
feedback mechanisms in female sheep. Behavioural effects such as oestrus exhibition due to
increased oestradiol concentrations are important during mating.
Luteal cellsLH–R
Granulosa cellsFSH–R
LH
InhibinOestradiol
-/+Hypothalamus
GnRH
Pituitary
Gland
LH
FSH
FSH Progesterone
Theca cellsLH–R
Ovary
Androstenedione
Follicle Corpus luteum
-/+
Corpus luteum +
Behavioural effects
13
2.13 The Oestrous Cycle
The length of the average oestrous cycle is 17 days with a modal range of 14 to 19 days in
most breeds of sheep (Fig. 6). As a matter of convenience, the beginning of the oestrous cycle
is counted from the time when the ewe has been marked by the male and is designated as Day
0. In a cyclic ewe, ovulation is preceded by oestrus behaviour (sexual receptivity) and
preovulatory LH surge which all these three phases occur within 72 hours (Martin et al.,
1986; Martin, Scaramuzzi & Lindsay, 1983; Oldham & Knight, 1979). The follicle that
ovulated is luteinized which becomes the corpus luteum, hence, called the luteal phase.
Figure 6: Oestrous cycle of the ewe.
Relationships among hormone levels, sexual receptivity and the time of ovulation,
demonstrating the key role played by PGA-F2α in inducing luteolysis. Adapted from Pineda
(2003b).
The progesterone avoids further ovulation in anticipation of an embryo implantation which its
concentration levels begin to rise rapidly and are detectable 3 days after ovulation. It reaches
maximum concentrations on Day 8 and remains elevated until Day 11 to 12 (Pineda, 2003b).
The cycle is therefore dominated by a luteal phase which lasts for 12 to 14 days and these
elevated progesterone concentrations keep the gonadotrophin at basal levels, particularly LH
(Pineda, 2003b). Inter-wave folliculogenesis occurs every 2 to 4 days in sheep and goats
during the luteal phase (de Castro et al., 1999; Ginther et al., 1995; Ginther & Kot, 1994;
Smeaton & Robertson, 1971). However, in most instances, the waves do not produce
ovulatory follicles and all follicles undergo atresia. Only the last wave of folliculogenesis is
14
vital as it emerges at the end of the luteal phase and the dominant follicles from this wave are
candidates for ovulation. The inter-wave folliculogenesis is self-regulated by oestradiol,
inhibin and progesterone in a negative feedback mechanism between the hypothalamus,
pituitary and the ovary (Scaramuzzi et al., 1993). Since oestradiol levels are secreted by the
large antral follicles, the blood levels of oestradiol remain low during most of the luteal
phase, but at times of inter-wave follicular growth, oestradiol levels do rise until atresia of
these non-ovulatory follicles occur (Pineda, 2003b).
In the absence of embryo implantation (no pregnancy), luteolysis is initiated as a
consequence of ovarian and posterior pituitary oxytocin release that stimulates endometrial
secretion of prostaglandin F2α (PGA-F2α) from the uterus (Pineda, 2003b). Prostaglandin F2
is a natural luteolysin (Porterfield, 2001b; Baird, 1984) and its luteolytic activity has been
described to: (i) constrict the utero-ovarian vessels causing ischemia (inadequate blood
supply) and starvation of the luteal cells; (ii) interfering with progesterone synthesis; (ii)
competing with LH for the receptor sites; and (iv) destructing the LH receptor sites
(Porterfield, 2001b). Progesterone levels in the peripheral blood decrease to less than 1.0
ng/mL (Pineda, 2003b). The alleviation of gonadotrophin suppression due to the low blood
levels of progesterone, oestradiol and inhibin, FSH synergises with LH to facilitate follicular
growth and oestradiol production from Day 14 to 16, called the follicular phase. The rapidly
rising levels of oestradiol towards the end of Day 16 induces oestrus behaviour in the ewe
which is shortly followed by preovulatory LH surge (Pineda, 2003b; Martin, 1984).
Ovulation within hours of LH surge signifies the start of the new cycle as a new corpus
luteum is formed and progesterone blood levels start to rise again.
2.14 “Male effect” for controlled breeding
There is sufficient evidence that demonstrated the effectiveness of teasing anoestrous ewes
elicit an acute physiological response when exposed to novel males (Martin, Oldham &
Lindsay, 1980; Oldham & Knight, 1979). Such male contact causes ewes to ovulate, but
oestrous cycle synchrony is not what we exactly want (Oldham, 1979). This knowledge has
shifted us into an era of looking at how nutritional supplementation could improve the ewe
response to the male effect. The following sections have therefore been revisited and will be
briefly discussed: i); the importance of isolating ewes from the males ii) ewe response to the
“male effect”; and, iii) our understanding so far with regards to the short cycle.
15
Our understanding of the aforementioned factors has made the use of male effect very
important as farmers can choose when to commence with the breeding season.
2.15 Isolation of ewes from males
There is abundance of evidence that previously isolated ewes respond spontaneously to the
introduction of males while continuous association causes females to familiarise with the
males and the “male effect” concept proves ineffective. The principle of pre-conditioning
disapproves of the farming system currently managed by subsistence farmers in Namibia. In
the Merino sheep that are also under a system of continuous association of sexes, the seasonal
breeding pattern of the breed is restricted and seems to be under light control (Schinckel,
1954b). Also, yearling ewes that continuously cohabited with males took on average 45 days
longer to start mating relative to when ewes were previously isolated (Notter, 1989).
In addition, continuous association can also mean that females show photoperiodic-driven
reproductive behaviour under such a system and their opportunistic strategies to coincide the
lambing and lactation with feed availability become out of phase (Martin et al., 2004).
Namibian breeds have a similar origin (Mediterranean) as the Australian breeds and the
“male effect” by isolating males and bucks from female sheep and goats should in theory
have the same effect, except that feeding strategies that are poorly applied in Namibian
conditions need to be improved on. In fact, isolation means that all the males are kept out of
sight of the females because the male odour, visual or sound can apparently mediate the
“male effect” (Gelez & Fabre-Nys, 2006; Pearce & Oldham, 1988).
2.16 Ewe response to the “male effect”
When previously isolated anoestrous ewes come in contact with males, ovulation within 3
days occurs that is preceded by an increased LH pulse frequencies and rapid LH surge which
is within 27 h (Oldham & Knight, 1979), 35 h (Knight, Peterson & Payne, 1979) and 36.7 h
(Ungerfeld et al., 2002). Ovulation varies from as few as 40 h (Oldham & Knight, 1979), 3
days (Martin et al., 1980; Knight et al., 1979), 5 days (Oldham, Boyes & Lindsay, 1984), to 6
days (Oldham, 1979; Schinckel, 1954a). These variations may be subject to (i) the nutritional
status of the animals at the time of male introduction; (ii) the breed genotype; (iii)
experimental methodology; (iv) time of season; (v) age of maturity: and (vi) environmental
factors. There is a two-peak cycle in one half of the group and one peak cycle in the other
half of the group (Oldham, 1979; Schinckel, 1954a). The two-peak cycle is a characteristic of
16
two LH surges within the space of 9 days, hence the short cycle. Ewes with a normal cycle
only have one LH surge within 72 h (Fig. 7).
Interestingly, short-term studies of the “male effect” on LH concentrations showed that after
24 h of exposure, the LH concentrations returned to basal levels similar to LH concentration
levels of the control group. On the other hand, FSH concentration levels fell within 2 hours of
male introduction and remain below basal levels of the control group for the remainder of the
experiment (Atkinson & Williamson, 1985).
All females are unable to exhibit manifest sexual receptivity at their first ovulation of male
introduction due to the prematurely regressing corpus luteum (Oldham & Pearce, 1984;
Martin, 1979; Oldham, 1979; Robinson, 1959; Schinckel, 1954b). Oestrus detection by males
and subsequent mating will only occur between 17 to 19 days and 23 to 25 days post male
introduction in the ewes experiencing the normal cycle and in ewes with the short cycle,
respectively (Fig. 7).
Figure 7: Two types of oestrous cycles induced in anovulatory ewes.
Response of anovulatory ewes after first ovulation within 72 h of male exposure. The first
normal cycle in one proportion of the ewe flock (top graph) and a short cycle of 6 days
(bottom graph) in the other proportion of the flock are both accompanied by silent heat at
both the first ovulation and, in ewes showing the short cycle, at the second ovulation. Ewes
showing a normal cycle will display their first oestrus on Day 19 and ewes having a short
cycle will display their first oestrus 6 days later. Adapted from (Martin et al. 1986).
17
2.17 The short cycle
At this stage, not much is known about what causes the short cycle in ewes. Though, it was
discovered that the inadequacy is not at the hypothalamic-pituitary axis level because
immunisation against androstenedione, to stimulate tonic secretion of LH (and not FSH) and
follicular activity does not prevent the short cycle nor does supplementation with oestradiol
(Martin, Scaramuzzi & Lindsay, 1981; Martin, 1979). This finding has shifted the attention to
the ovary and we now know that the follicle maturation does not complete and that results in
a poor quality corpus luteum which only lasts for 9 to 10 days after male-induced second
ovulation .
The period of delay from male introduction to LH surge is important to allow follicular
growth and it was suggested the possibility of a relationship between the LH surge and the
type of corpus luteum formed (Pearce, Martin & Oldham, 1985). Importantly, priming ewes
with progesterone does not block the LH synthesis and secretion, but it only delays its surge
(Martin et al., 1983). As this will present a long follicular phase, sufficient time is afforded to
synchronise follicle growth with other endocrine processes (Martin et al. 1986). Therefore,
the “window of opportunity” is a prerequisite, allowing FSH concentrations to remain
elevated so that gonadotrophin-dependent follicles continue to grow into ovulatory follicles.
Unfortunately, to achieve this, using natural methods to improve reproductive performance in
animals is a tall order bacause ovarian activity or the state of follicles varies from ewe to ewe
leading to a high degree of randomness.
2.18 Nutrition
Over the past 90 years, many workers had made numerous advances in animal nutrition and it
is undoubtedly the most important environmental factor to improve the reproductive
performance in small ruminants (Smith & Stewart, 1990; Hafez, 1952). Nutrients boost
programming and expression of metabolic pathways that enable animals to achieve their
genetic potential for reproduction. In other words, the feeding regimes will have a long-term
or permanent change in the subsequent reproductive performance (Lucas, 1992). The
suggestion was that poor nutrition may have consequences on the number of gonadotrophin-
dependent follicles (Scaramuzzi et al., 1993). Thus, changing levels of nutrition at a critical
or sensitive period of reproduction, are essential and its effects in the efficiency of most
stages of reproductive processes are by far the most important on ovulation rate (Smith,
1985). Research on nutrition and ruminant fertility concentrates from the whole animal
responses to particular and complex functions of cellular and molecular processes that control
18
gamete production, ovulation, fertilization, embryo development and survival, conceptus
implantation and growth (Robinson et al., 2006; Holst, Killeen & Cullis, 1986).
2.18.1 Types of nutrition
Two types of nutritional effects, dynamic and static effects, were invented where increased
ovulation rate is associated with change in bodyweight (Coop, 1966; Coop, 1962). In
contrast, Lindsay et al., 1975 discovered the acute effect using lupins in short-term nutritional
strategies to significantly increase ovulation rate. The three effects of nutrition have been
adequately explained by Smith & Stewart, 1990 and will be briefly discussed for the purpose
of the choice for this thesis.
2.18.1.1 Acute effect
This approach uses short-term feeding strategies to increase ovulation rate. It is so acute that
increased ovulation rate becomes apparent long before the changes in body condition have
their effects (Fig. 8). For example, when ewes are fed lupin supplements: (i) plasma glucose,
acetate and propionate increase; (ii) entry rates of glucose, acetate and carbon dioxide into the
cells are accelerated; and (iii) glucose oxidation increases (Teleni et al., 1989a). On the other
hand, intravenous glucose infusion only caused increased glucose entry rates at the highest
infusion rate of 46.8 mmoles h-1 (cf. 31 mmoles h-1 due to lupins) (Teleni et al., 1989a) and
60 – 65 mmoles h-1 for 5 days in the late luteal phase of the oestrous cycle (Downing, Joss &
Scaramuzzi, 1995). The energy levels provided by lupins prove adequate to increase
ovulation rate compared to the intravenous infusion of glucose.
Supplementing ewes with lupins for as few as 4 and 6 days prior to ovulation can
significantly increase the ovulation rate (Stewart & Oldham, 1986; Oldham & Lindsay,
1984). This was in agreement when ovulatory response to lupin grain supplements was
initiated near the time of luteolysis (Nottle, Seamark & Setchell, 1990). Short-term
supplementation from Days 8 to 14 of the oestrous cycle increases ovulation rate in ewes
with a moderate to high body condition due to acute changes in the blood glucose, insulin and
leptin concentrations (Viñoles et al., 2003; Viñoles, 2003). These critical time ranges of
short-term nutritional supplement close to luteolysis is when the ovulatory wave emerges
(Viñoles, 2003). A lupin diet with protein (34%) and metabolizable energy contents 13.7
MJ/kg (Milton, 2001) can induce high ovulation rate which promotes the consumption of
increased digestible energy presumably mediated via biological pathways associated with the
19
synthesis and utilisation of glucose for reproduction (Leury, Murray & Rowe, 1990; Teleni et
al., 1989b; Murray & Rowe, 1984).
47
48
Nutritional supplement
“Acute” “Dynamic” “Static”
0 2 4 6 8 10 12 14 16 18 20
1.0
1.5
2.0
44
45
46
Days after the start of feeding
Ovulation
rateBody weight
(kg)
Undetectable
Figure 8: Acute effect of feeding.
The result leads to positive energy balance which, in turn, increases leptin and insulin
concentrations in the blood subsequently increasing the glucose uptake by the cells. These
effects seem to have a direct effect on the ovary which are linked to increased folliculogenesis
and ovulation rate (arrow indicating acute). The arrow designated undetectable illustrates
that change in body weight is imperceptible at the equivalent time when increased
folliculogenesis and ovulation rate are detected. Adapted from Scaramuzzi et al. (2006).
2.18.1.2 Static effect
Static effect is the body condition of ewes within the same genotype and female size that
enter the breeding season which is indicative of a stable metabolic status. Depending on their
different levels of body condition, ewes will have different ovulation rates. Body condition
affected the size of the large follicles (≥4 mm diameter) during the late luteal and follicular
phases (Rhind & McNeilly, 1986) and ewes in high body condition had higher FSH and low
oestradiol (E2) concentrations during the follicular phase than ewes in a low body condition
(Viñoles et al. 2002). Rhind & McNeilly, 1986, however, found that the difference in mean
FSH concentrations during the follicular phase was unclear but concluded that there is a
greater suppression of FSH in ewes in good condition. They indicated that such suppression
is the result of higher oestrogen and inhibin concentrations synthesised by the greater number
of large oestrogenic follicles present. This is a possible explanation because the serum
20
concentrations of FSH decline at maximum follicular growth between 2 to 3 days prior to
oestrus (L'Hermite et al., 1972). It is the time when large oestrogenic follicles exert a
negative feedback on the hypothalamic-hypophyseal axis to restrict the further growth of
other developing follicles (Baird et al., 1975).
The “static effect” of nutrition on ovulation rate in ewes in a higher average body condition
of 2.86 had a higher ovulation rate (1.8 v 0.9) and more large follicles with ≥4 mm in
diameter than ewes in a lower average body condition of 1.84, but the numbers of small
follicles between 1 mm – 4 mm in diameter were similar in both groups (Rhind et al., 1989;
Rhind & McNeilly, 1986). This was further supported when ewes with high body condition
(4.1) had high levels of FSH concentration during the follicular phase, thus, allowing a longer
period of follicle recruitment (Viñoles et al., 2002).
Ewes with a low live weight of 41.6 kg compared to heavier ewes of 56.5 kg: (i) had low
ovulation rate; (ii) came into oestrous later upon the withdrawal of the sponges; (iii) had more
follicles ≥2 mm in diameter in late luteal phase; (iv) had less number of follicles that were
recruited early in the follicular phase; and (v) a high intensity of selection through atresia
(Xu, McDonald & McCutcheon, 1989).
2.18.2 Acute versus Static Effect
The diagrams in Scaramuzzi et al., 2006; Viñoles et al., 2005 illustrate the comparisons of
immediate and static effects on hormones, glucose, insulin and leptin concentrations that rise
rapidly with peak levels experienced on day three after supplementation commenced. Static
effect is responsible for: (1) increased FSH concentrations mediated via the hypothalamus;
(2) development of more follicles; and, (3) decreased oestradiol (E2) concentrations. At the
ovarian level, FSH stimulates the development of more follicles which eventually promotes
an increase in ovulation rate. During this period of high FSH levels, E2 production by the
follicles is low which is reported to be the result of high leptin concentrations that inhibit
steroidogenesis in ewes with high body condition. The lower oestradiol concentrations reduce
negative feedback at the hypothalamus and pituitary gland that lead to increased circulating
FSH concentrations.
Contrary to the static effect, the immediate effect of nutritional supplement promotes acute
responses of: (1) increased glucose concentrations; (2) increased insulin concentrations; (3)
increased leptin concentrations; and, (4) therefore stimulation of follicular growth. This is
indicative of positive energy balance and these changes increase folliculogenesis and
21
ovulation rate in sheep as consequence of the direct effect on the ovary (Scaramuzzi et al.,
2006).
2.18.2.1 Dynamic effect
Ewes at any given live weight have different ovulation rates caused by the varying levels of
body condition preceded by a period of supplementation (e.g. weeks) and this is known as the
‘dynamic effect’. Providing extra feeding for several weeks has proved to increase
reproductive performance in breeding stock (Kiyma et al., 2004; Adams et al., 1997; Nottle
et al., 1997; Blache et al., 1996; Boukhliq, Adams & Martin, 1996; Nottle et al., 1990;
Stewart & Oldham, 1986; Lightfoot & Marshall, 1976; Lightfoot, Marshall & Croker, 1976;
Knight, Oldham & Lindsay, 1975). However, timing of feeding and type of feeding appear to
play a role to elicit increased ovulation rate. It took approximately three weeks to observe
ovulation rate responses when high pasture allowances were fed to ewes (Smith, Jagusch &
Farquhar, 1983). Coop, 1966 demonstrated though that ewes need to be fed high energy
supplement for one entire oestrous cycle if increased ovulation rate is to be expected. This
was supported by Dufour & Matton, 1977 who found that feeding such diet for less than one
oestrous cycle does not result in increased ovulation rate. The shortened supplementation can
only be achieved when nutrients, e.g. glucose and acetate are infused for nine days to produce
an energy intake equivalent to that of a maintenance diet plus 750 g of lupins (Teleni et al.,
1989b; Teleni & Rowe, 1986). It is almost inconceivable to suggest that farmers have to
supplement for the entire 17 days of the oestrous cycle to achieve high ovulation rates.
Moreover, although high ovulation rate is achieved with supplementation, it does not
necessarily mean high conception rate, thus, feeding animals for such a long period may not
produce good economical returns.
2.19 Nutrition to increase ovulation rate
The physiological processes that control folliculogenesis will produce in majority of breeds
between 1 and 2 dominant follicles (Scaramuzzi & Radford, 1983) and, in other prolific
breeds such as Booroola Merino, Dahman, Finn and Romanov, as many as 3 or more
dominant follicles that are destined to ovulate (Scaramuzzi & Radford, 1983; Lahlou-Kassi &
Marie, 1981; Bindon et al., 1979; Bradford, Quirke & Hart, 1971). This is termed ovulation
rate which determines fecundity (e.g. litter size) in breeds. Ovulation rate is defined as the
number of ovulations in a flock divided by the number of ewes ovulating which is a
characteristic that displays a considerable degree of genetic variation. Ovulation rate is
determined by counting the number of corpora lutea appearing on both ovaries of each ewe.
22
The ovulation rate, whether it is single or multiple ovulations, is apparently driven by several
factors that work in harmony and they are: 1) the number of immediately available
gonadotrophin-dependent follicles to be transformed into gonadotrophin-responsive follicles;
2) the individual requirement for FSH by gonadotrophin-dependent follicles; and, 3) the
degree of response of hypothalamic-pituitary axis to inhibin and oestradiol inhibitory effects
(Scaramuzzi et al., 1993). The next section will concentrate on the influence of nutrition on
ovulation with reference to the types of nutritional effects.
2.20 Nutrition to improve response to the male effect
Comparing the results of various experiments with lupin supplements to ewes for 35 days
before mating and another 18 days during mating showed significant increases in ovulation
rate, lambing and twinning rate. Different rates of lupins (0, 125, 250 and ad libitum
g/ewe/day) and different durations of feeding (0, 7, and 14 days prior to joining) showed no
significant difference when feeding ewes for 7 or 14 days, but it was higher than ewes that
received supplementation on Day 0 of mating (Lightfoot et al., 1976). Interestingly, the rate
of feeding at 250g/ewe/per day for all three durations had higher ovulation rate than feeding
at ad libitum. In addition, supplements with 250g/ewe/per day of sweet lupin for 14 days
before mating until Day 17 of mating had a variation of -14 to +21 % in terms of lambs born
(Croker, Johns & Johnson, 1985). Importantly, supplementing at 500g/ewe/per day did not
alleviate the problem of variable response. This supported Lightfoot et al., 1976 who
suggested that there is perhaps a threshold at which nutrition stops influencing ovulation rate.
In goats, supplementary feeding for seven days prior to buck exposure increased ovulation
rate at first male-induced ovulation in the treated group compared to the control group, but
nutrition had no effect in the subsequent ovulation (De Santiago-Miramontes et al., 2007).
They further found that the proportion of does with the short cycle was higher in the treated
group than the control group.
2.21 Summary
The literature review has highlighted the importance of nutritional supplementation to the
overall reproductive performance in small ruminants. Many reporters have demonstrated the
synergistic effects of nutrition and the “male effect” to induce ovulation in a higher
proportion of anovular ewes to increase ovulation rate in higher proportion of ewes with twin
ovulations. The use of the “male effect” is an environmentally friendly and non-hormonal
tool to control the breeding season. This allows farmers to adopt strategies to maximise on
23
the reproductive performance of the breeding stock. For example, a farmer can change the
breeding season from autumn to spring mating so pregnancy and lactation overlap with the
rainy season. Although this may lead to low lambing percentages the benefit of lamb survival
and possibly heavier lambs as a result of sufficient food availability outweighs most factors
of autumn mating.
Improved responses to the “male effect” is possible with short-term nutritional
supplementation as opposed to prolonged feeding strategies. Despite this, the short-term
nutritional supplements are easily feasible by studying the ovarian activity with laparoscopy
and ultrasound techniques during the oestrous cycle only and not during an otherwise
quiescent ovary. For example, nutritional supplements are applied at the emergence of the
last follicular wave (Days 8 to 13 of the cycle), just before luteolysis, to take advantage of the
time of the synchronous growth of the antral follicles under the influence of FSH. More work
needs to be conducted in anoestrous ewes to fully understand when it is the best time to
supplement ewes to stimulate follicle development. Much of the research is now focussed on
the effects of external and internal inputs that converge into a common pathway to induce
ovulation in sheep and goats. Further understanding of these pathways could help to
manipulate nutrition and the “male effect” strategies, especially with regard to the short
cycle.
24
CHAPTER 3
3.1 General Methods and Materials
This experiment was carried out in accordance with the Australian Code of Practice for the
Care and Use of Animals for Scientific Purposes (8th Edition, 2013) and was approved by the
Animal Ethics Committee of The University of Western Australia under RA 03/100/596.
3.1.1 Area of study
Both experiments were conducted at Allandale Farm, a field station of The University of
Western Australia (UWA) that lies between 110º and 120º W and 30º and 40º S north, 60 km
northeast of Perth, Western Australia. The farm has mixed-age Merino ewes ranging from 3
to 10 years old, but in this experiment only ewes in the age range 3-6 years old were selected.
Experiment 1 was begun in mid-January, the start of the normal breeding period at the farm,
just before the onset of the normal breeding season for Merinos. Experiment 2 was carried
out during the non-breeding season at the end of spring (September-November) and the
beginning of the summer (December-February). The climate is Mediterranean and the studies
were conducted under natural light. In both experiments, animals were kept in paddocks.
Grazing is mainly extensive with occasional feeding of hay to alleviate nutritional stress
during the dry spells of the year (late October to early May). Rainfall normally starts late
autumn and carries through winter until early spring.
Experimental animals
In all experiments, Merino sheep were used to define the effects of lupin grain and the ‘male
effect’ on reproductive performance. All the animals selected for the experiments were
sexually experienced and had lambed at least once.
3.2 Experiment 1
Before the experiment commenced, all the ewes were running on dry summer pastures and
were fed hay ad libitum. Transrectal ultrasonography using a real-time B-mode machine
(Aloka SSD500, Aloka Co. Ltd., Tokyo, Japan) equipped with a 7.5 MHz linear array
Transducer was performed to determine the proportion of the flock that was cyclic (corpora
lutea on either or both ovaries) and to allow selection of acyclic ewes (no corpora lutea). The
acyclic ewes were allocated among three treatment groups; some were subsequently removed
when retrospective analysis of oestrus observations suggested that they were cyclic but not
detected by ultrasound examination. Prior to randomised allocation into three groups, the
ewes were weighed and condition-scored (scale 1 to 5, with 1 being emaciated, 2.5 medium
25
fat and 5 very fat; Russel, Doney & Gunn, 1969; Jefferies, 1961). The three experimental
groups were: Control (n = 32; not fed any supplement); Lupin-supplemented before male
introduction (L-Pre; n = 23); Lupin-supplemented after male introduction (L-Post; n = 30).
Experiment 1 was split into 2 parts.
Part 1
Prior to male introduction, the L-Pre group was run separately and fed at a rate of 500 g lupin
grain per ewe per day for 6 days, while the Control and L-Post groups were run together. All
groups were combined on the final day of L-Pre supplementation and testosterone-treated
wethers fitted with harnesses and crayons were introduced. Any ewes that showed oestrus
within the 14 days from introduction of the wethers were removed from the experiment. The
end of 14 days was also the start of the breeding programme on the farm and the wethers
were replaced with testis-intact rams fitted with different crayon colours at a 6% ratio. The
colours of the crayons on the rams were changed on Days 21 and 28.
Part 2
The L-Post group was separated from the Control and L-Pre groups and they were fed lupin
grain at 500 g per ewe per day for 12 days from Days 11 to 22 of the oestrous cycle, which
was after male introduction. On Day 55, trans-abdominal (3.5 MHz linear-array transducer)
ultrasonography was used to determine pregnancy and to count the number of foetuses per
pregnant ewe.
3.3 Experiment 2
The Merino ewes (n = 112) had all lambed once before, but not in the preceding lambing
season to ensure that ewes were anovulatory. As in Experiment 1, ultrasound was used to
separate cyclic from acyclic ewes, and the acyclic ewes were allocated among two equal-
sized groups: Control (n = 29) and L-Pre (n = 29). Throughout the experiment, ewes were
weighed and condition scored every 7 seven days, as described in Experiment 1.
The two treatment groups were run in separate paddocks. The Control group was fed hay ad
libitum on dry pastures; the L-Pre group was fed 500 g of lupin grain per ewe per day prior to
male introduction, from Day –5 to Day 0. At the end of nutritional supplementation, on Day
6, the groups were combined and vasectomized rams fitted with harnesses and crayons, at a
ratio of 1:10, were introduced.
26
Oestrus observations
In Experiment 1, oestrus distribution was determined by recording marked ewes in the
morning and late afternoon every day, from Day 16 to Day 26 after initiation of the ‘male
effect’. In Experiment 2, all ewes marked within the first 14 days after male introduction
were recorded, but only data from ewes with distinct crayon marks on the rumps were
retained for analysis. Crayons were replaced on Day 14 after ram introduction and then every
7 days until Day 28. Oestrus observations were performed every day between Days 16 and
26.
Examination of the ovaries
All experimental animals were kept overnight in the yards with no feed or water. Ovulation
rate was determined by counting the number of corpora lutea on both ovaries per ewe
ovulating by directly viewing the ovaries with a laparoscope (Oldham and Lindsay 1980).
Five minutes before laparoscopy was started, 2 mg xylazine was injected intramuscularly for
partial sedation. Ewes were placed in a crate in a vertical, dorsal recumbent position. At the
end of the laparoscopy, 5 mL of benacillin was injected intramuscularly and the wound was
sprayed with vetericyn, all supplied by the veterinarian who performed the laparoscopy for
this experiment.
Statistical analyses
We have based the analysis of treatment effects only on data collected from ewes that were
deemed to have responded to the ‘male effect’, as evidenced by the occurrence of oestrus
between Days 16 and 26. The number of corpora lutea per ewe ovulating (ovulation rate) was
analysed using Chi-squared tests (χ2) in a 2 x 2 contingency table. Oestrus distributions for
short versus normal cycles were determined using histograms with fits and groups in the
Minitab 14 programme.
27
CHAPTER 4
4.1 Introduction
Sheep farmers place a lot of emphasis on maximising lamb output per ewe reproductive
lifespan and a major contributor to lamb output is the ovulation rate of the ewe, the ultimate
factor that determines fecundity (litter size). The upper limit for ovulation rate is set by
genotype, but the degree to which a ewe can express her genetic potential is determined
largely by nutrition. For decades, researchers have been intrigued by the mechanisms through
which nutrition influences ovulation rate and, consequently, how nutrition can be optimally
utilised to increase fecundity. Most of the research on sheep and goats has used a
combination of ‘medium- and long-term’ feeding regimes (2 to 8 weeks), before and during
mating, to boost energy levels (Coop, 1966; Findlay & Cumming, 1976; Fletcher, 1981;
Lightfoot & Marshall, 1976; Lightfoot, Marshall & Croker, 1976; McInnes & Smith, 1966;
Molle et al., 1995; Molle et al., 1997; Radford, Donegan & Scaramuzzi, 1983; Smith,
Jagusch & Farquhar, 1983). However, in extensive and subsistence production systems, the
costs of long periods of high-level feeding are prohibitive except when pasture is abundant,
so it is not economically viable to feed animals so they can reach their genetic potential at all
times of the year. This means that the manipulation of nutrition is restricted to short-term
supplementation.
Research has shown that, if they are carefully timed, short-term supplementation can enable
animals to achieve their genetic potential by directly stimulating the metabolic processes that
control ovulation (Smith & Stewart, 1990). The ultimate refinement of this concept is ‘focus
feeding’ in which supplements fed for only a few days are used to increase ovulation rates
(Nottle et al., 1992; Nottle, Seamark & Setchell, 1990; Oldham & Lindsay, 1984; Viñoles et
al., 2003).
Short-term nutritional supplements, such as lupin grain (Lupinus angustifolius), and related
nutritional stimuli, such as glucose infusion, stimulate follicle development by promoting
FSH-stimulated intracellular signalling pathways in the granulosa cells, yet somehow reduce
the secretion of oestradiol. This leads to changes in the balance of the negative feedback
loops in the hypothalamo-pituitary-ovarian axis that control gonadotrophin secretion, so that
FSH concentrations can be sustained during the follicular phase of the oestrous cycle even
when more follicles are growing and developing (Scaramuzzi et al., 2006; Somchit et al.,
2007; Viñoles et al., 2005).
28
The general consensus is that the critical time during which nutrition can influence ovulation
is around the end of the 12-day luteal phase (Nottle et al., 1990; Stewart & Oldham, 1986)
when the last wave of follicular development culminates in the production of ovulatory
follicles (Viñoles et al., 2003a). For such accurate timing of supplementation, the time of the
next expected ovulation needs to be known. Until now, this has been feasible through the use
of exogenous hormones (progestagen, prostaglandin, gonadotrophin) to control the cycle,
although these treatments are also too expensive or impractical for subsistence and extensive
production systems. In anoestrous ewes, an alternative that is theoretically feasible and
affordable is the induction of ovulation with the ‘ram effect’. However, there are three
problems with the ram effect:
i) There is considerable variation among individuals, flocks and genotypes in the
responsiveness of the ewes to the ram effect (Martin et al., 1986);
ii) On average, after ram-induced ovulation, 50% of the ewes in a flock experience a
short cycle and the other 50% a normal cycle, so cycle synchrony is far from perfect
(Oldham, 1979; Schinckel, 1954);
iii) The ‘ram effect’ itself contributes to variation in ovulation rate (Cognie et al., 1980)
with unknown outcomes under variations in short-term nutrition.
All three of these problems can be linked to the processes controlling the growth and
development of the follicles in the ovary. Follicular waves continue throughout anoestrus,
with follicles emerging, developing and becoming atretic, but the rate of development may be
dampened in deeply anoestrous ewes compared to ewes with a shallow anoestrus. The short
luteal phase is reported to be caused by either poor transformation of the ovulating follicles
into corpora lutea, or by premature regression of the corpora lutea (Martin et al., 1986). In
both cases, the underlying cause may be the status of the follicles at the time of ram
introduction. Finally, the number of follicles that ovulate (ovulation rate) at the ram-induced
ovulation is also determined by variations in the processes of folliculogenesis (Scaramuzzi et
al., 1993).
Because nutrition exerts a wide range of largely beneficial effects on folliculogenesis,
perhaps it could be used to improve the outcomes of the ram effect.
Therefore, in the present study, we tested whether a nutritional supplement, fed for 6 days
before the introduction of the males will improve the outcomes for the male-induced
ovulation. In the subsequent male-induced oestrous cycle, we also tested whether
supplementation for 12 days in the late luteal and follicular phases would affect ovulation rate
29
at the first oestrus (second/third ovulation), based on the knowledge that some farmers in
Western Australia apply this nutritional strategy during the breeding season.
The specific hypotheses tested were:
a) Nutritional supplementation for six days before male introduction will increase
number of ewes that ovulate in response to the ‘male effect’;
b) Nutritional supplementation for six days before male introduction will prevent
short cycles;
c) Nutritional supplementation during the male-induced cycle will increase ovulation
rate at the first oestrus.
4.2 Materials and Methods
The ewes selected for the experiment were part of the main breeding flock at Allandale, the
farm of the University of Western Australia. The experiment was begun in mid-January, the
start of the normal breeding period at the farm, just before the onset of the normal breeding
season for Merinos in the southern hemisphere (Pearce and Oldham 1988) and ended on
April 30. Before the experiment commenced, all the ewes were on dry summer pastures and
were fed hay ad libitum. Transrectal ultrasound was performed on 60 ewes, eight days before
the experiment started, to determine the proportion of the flock that was cyclic and to allow
selection of ewes deemed as acyclic. Cyclic ewes were described as such if they had corpora
lutea on either or both ovaries whereas acyclic ewes had no corpora lutea. The acyclic ewes
were allocated among three treatment groups and then some ewes were subsequently deleted
when retrospective analysis of oestrus observations suggested that they were cyclic but not
detected by ultrasound examination. The final three groups were: Control (n = 32) not fed
any supplement; Lupin-supplemented before male introduction (L-Pre; n = 23); Lupin-
supplemented after male introduction (L-Post; n = 30).
The experiment consisted of two parts: in Part 1, we compared the Control and L-Pre groups,
and Part 2 we compared the Control and L-Post groups. Initially, the Control and L-Post
groups were run together, with L-Pre group in a separate field. In all fields, the animals were
fed hay ad libitum on dry pastures. All ewes were weighed and condition scored every seven
days. Ovulation was induced by the introduction of wethers (6 per 100 ewes) that had
received three subcutaneous injections of 2 mL of testosterone at 7-day intervals.
30
4.3 Part 1
For 6 days, the L-Pre ewes were fed as a group with 500 g lupin grain per ewe daily (Fig. 9).
On the final day of L-Pre supplementation (= Day 0 of the male-induced cycles), all three
groups were placed in the same field and testosterone-treated wethers were introduced. The
wethers had harnesses with crayons to mark ewes that showed oestrus. The ewes marked
within the first 14 days were removed from the experiment as retrospective calculation of the
timing of oestrus indicated that they were cyclic prior to the wether treatment. Day 14 was
also start of the normal breeding programme on the farm and the wethers were replaced with
entire rams, carrying a different colour crayon, at the same ratio (6%). On Days 21 and 28,
the colours of the crayons carried by the rams were changed so we could detect the week
within which the ewes came into oestrus.
castrated males, testosterone -
treated, with green markers
14
remove marked ewes, change
marker colour, replace castrates
with fertile rams
Ultrasound
for anoestrus
-8 28
marker
colour
20 21
marker
colour
30
Laparoscopy
0
6-day
supplementation
-5 11 22
12-day supplementation
15
Laparoscopy
Figure 9: A schematic illustration of the experimental design.
Laparoscopy procedure
Ovulation was determined by counting the number of corpora lutea on both ovaries of each
ewe on Day 15 in the L-Pre and C groups only, by directly viewing the ovaries with a
laparoscope (Oldham and Lindsay 1980).
31
4.4 Part 2
The L-Post group underwent the same protocol as above except the feeding regime (lupin
grain at 500 g per ewe daily) ran for 12 days, from Day 11 to Day 22 (inclusive) after male
introduction. The L-Post group was run separately from the L-Pre and Control ewes during
the treatment period, after which all three groups were again recombined. In relation to the
two potential outcomes of male-induced ovulation, the timing of L-Post supplementation
would have been either, a) from the second ovulation of the male-induced ovulatory cycle for
the ewes having a normal cycle only, or b) from the third male-induced ovulatory cycle for
the ewes experiencing the short cycle. The timing of this feeding regime was in anticipation
when the supplement would have an effect on the ovulation.
Oestrus distribution was determined by recording marked ewes in the morning and late
afternoon every day, from Day 16 to Day 26 after the ‘male effect’. On Day 32 of the
experiment, laparoscopy was again used to determine ovulation rate. On Day 55, ultrasound
was used to detect pregnant ewes and to count the number of foetuses per pregnant ewe.
Statistical Analysis
We have based the analysis of treatment effects only on data collected from ewes that had
most probably responded to the ‘male effect’, as evidenced by the occurrence of oestrus
between Days 19 and 29. The number of corpora lutea per ewe ovulating (ovulation rate) was
analysed using Chi-squared tests (χ2) in a 2 x 2 contingency table. Oestrus distributions for
short versus normal cycles were determined using histograms with fits and groups in the
Minitab 15 programme.
4.5 Results
Live weight
The live weight data are presented in Figure 10. The weights did not differ among the three
groups at the beginning or at the end of the experimental period (P > 0.05). There were
similar trends of slight weight gains of 0.91 g, 0.61 g and 0.76 g for Control, L-Pre and L-
Post, respectively. The L-Post group tended to be heavier throughout the experiment than
Control and L-Pre groups.
32
Figure 10: The mean live weights of the ewes.
Weighing was done prior to and at the end of the experimental period, in the L-Pre (short
broken lines), L-Post (solid line) and Control (long broken lines) groups. There were no
significant differences (P > 0.05).
Ovulation and oestrus
The data for oestrus, ovulation and ovulation rate, and the proportions of ewes with short and
normal cycles after the ram-induced ovulation, are shown in Table 1. In all three groups,
almost all ewes ovulated so there was no effect of treatment on the proportion of ewes
responding to the male effect. The percentage of ewes ovulating was closely followed by the
percentage of ewes showing oestrus and more ewes ovulated than displayed oestrus in all
three groups.
Ovulation Rate
The ovulation rate tended to be higher in the L-Pre group than in the Control (P > 0.05) at the
first ovulation and was significantly higher in the L-Post group than in the Control (P < 0.05)
at laparoscopy on Day 32, at the second ovulation for ewes with a normal cycle and at third
ovulation for ewes with short and normal cycle.
43
44
45
46
47
48
49
50
51
1 2
Liv
e w
eigh
t o
f ew
es (
kg)
Weighing of ewes prior to and after the experimental period
33
Oestrus Distribution
Figure 11 shows the distribution of oestrus for each group with the bars representing the
frequency of ewes marked on each day of observation.
Table 1: Reproductive variables:
Reproductive variables in ewes induced to ovulate using the “male effect” at the onset of the
breeding season. Data were collected at laparoscopy on Day 15 for L-Pre and Day 30 for L-
Post. Percentages are based on n; different superscripts indicate significance (P < 0.05).
Control L-Pre L-Post
n 32 23 30
Ewes ovulating (%) 94 100 100
Ovulation Rate 1.37ª 1.48ª 1.66b
Short cycle (%) 34a 39a 23b
Normal cycle (%) 41a 48a 67b
Oestrus (%) 75 87 90
Figure 11: Oestrus distribution curves in ewes.
Distribution curves illustrating the number of ewes with a normal cycle (clear bars) and a
short plus a normal cycle (shaded bars) and days of oestrus occurrence in a) Control, b) L-
Pre and c) L-Post groups.
34
Following male-induced ovulation, the pattern of oestrus was similar for L-Pre and Control
(P < 0.05). In all groups, more ewes were marked between Days 19 and 20 than between
Days 21 and 29, indicating a concentration of normal cycles.
Oestrous Cycle
Figure 4.3 shows the percentage of ewes in each group that expressed normal and short
cycles. The proportion of ewes with the normal cycle was higher than the number of ewes
with the short cycles in the L-Post group (P < 0.05). This difference was not seen in the C and
L-Pre (P < 0.05). Consequently, 67% of the ewes experiencing a normal cycle in L-Post
group were significantly more than the ewes with a normal cycle in C and L-Pre groups (P <
0.05).
Pregnancy Rate
The pregnancy data are shown in Table 2. Pregnancy rates did not differ among the groups.
There were significantly more twin (and thus less single) foetuses in L-Post than in the other
two groups.
Table 2: Singles, twins and overall pregnancy rates:
Data from ultrasound pregnancy diagnosis on Day 50 after male-induced ovulation.
Different superscripts indicate significant differences (P < 0.05).
Control L-Pre L-Post
n 32 23 30
Singles (%) 74 63 45
Twins (%) 26a 37a 55b
Pregnant (%) 72 83 73
35
4.6 Discussion
The use of nutrition to increase the number of ewes ovulating was effectively not tested
because ovulation was observed in all ewes in the L-Pre and L-Post groups and 94% of
Control ewes, suggesting a strong response to the male effect and perhaps a major
contribution from spontaneous ovulations. The second of these possibilities is supported by
the fact that 10% of ewes in the flock were observed to be cycling when the experimental
animals were selected just before the experiment. The time of the season probably played a
major role – the study was begun around the expected onset of the normal, photoperiod-
induced breeding season when we would expect there to be an increasing number of ewes
becoming spontaneously cyclic. These observations indicate that the Merinos on Allandale
Farm have a shallower anoestrus than expected, and are thus highly sensitive to the male
effect. Other observations of this flock support this contention (P Hawken, T Jorre de St Jorre
2008, pers. Comm.. 2008), suggesting that a reduction in seasonality has developed over
recent years in the University flock. In previous studies with Merino ewes, generally, less
than 100% ovulated in response to the ram effect in the absence of lupin supplementation
(Fletcher & Geytenbeek, 1970; Pearce & Oldham, 1988; Wheeler & Land, 1977). In breeds
that are more seasonal than Merinos, the response tends to be lower (review: Martin et al
1986). In these genotypes, it would be worthwhile testing again the hypothesis that nutritional
supplements can improve the ability of the ram effect to induce ovulation.
On the other hand, the nutritional supplementations appear to have stimulated follicle growth,
as evidenced by increases in ovulation rate. The effect of supplementations on ovulation rate
was thought to be more apparent when ewes had previously been kept on poor quality pasture
(Gherardi & Lindsay, 1982). This certainly applies to the current experiment because the
ewes were run on dry summer pasture and, although they were given hay ad libitum, it was
poor quality. Therefore, even with small numbers of experimental animals, we observed a
response to the second period of supplementation as natural seasonal grazing conditions were
deteriorating.
The hypothesis that a nutritional supplement could be used to prevent short cycles in ewes
responding to the ‘male effect’ was rejected because 39% of L-Pre and 41% of Control ewes
experienced short cycles. However, we need to be cautious in this interpretation because the
power of our experimental design was limited by feeding the ewes in groups rather than in
individual pens. This could pose a problem because there can be large differences among
36
ewes in the intake of lupins. In addition, group feeding behaviour, leading interactions among
the animals, could increase this variation. At the extremes of this variation, we would expect
major differences in the way the follicles respond and grow to nutritional stimulus (Murray &
Rowe, 1984). On the other hand, we did detect significant responses to the second
supplementation for ovulation rate, so folliculogenesis was affected by the treatment. At this
stage, we therefore conclude that a nutritional supplementation is not likely to reduce the
likelihood of induction of short-life-span corpora lutea.
A smaller proportion (23%) of ewes in the L-Post group had a short cycle, compared to
around 50% in the other groups. This is probably a chance observation because the
supplement for L-Post ewes was not begun until 11 days after the ‘male effect’, by which
time the distribution among short and normal cycles should have been determined. It is
feasible that the 12-day nutritional supplementation may have facilitated an extension of the
follicular phase of the oestrous cycle. As an a posteriori observation, this needs to be tested
again as an a priori hypothesis.
The data for oestrus distribution showed that, in all three groups, the ewes that experienced a
normal cycle following male-induced ovulation came into oestrus in a brief period, mostly
over Days 19 and 20. In contrast, there was a considerable variability among the ewes
experiencing a short cycle, with oestrus exhibited over a 9-day period (Days 21-29). These
ewes could not have been cyclic before the ‘ram effect’ as they were not marked in the first
14 days after male introduction. In all groups, small proportions of ewes ovulated but failed
to show oestrus, or showed oestrus but apparently did not ovulate, or conceived without being
detected in oestrus. Such variations in the response to the ‘male effect’ have been previously
reported for Corriedale ewes (Ungerfeld et al., 2002) although it is difficult to rule out
technical problems such as errors in detection of oestrus. Interestingly, 24-hourly repeated
short-term exposure of ewes to rams in temperate regions led to improved synchrony of the
oestrous cycle, thus a compact and earlier lambing. This suggests that continuous presence of
rams is not essential, especially during the transition of the anoestrous into the breeding
season (Hawken, Evans & Beard, 2007).
Nutritional supplementation for 12 days during the oestrous cycle improved reproductive
performance, as evidenced by the increased percentage of twin foetuses observed at scanning.
This supports the conclusions drawn for ovulation rate. Therefore, an additional period of
37
feeding to the 6-day nutritional supplementation before the ‘male effect’ is recommended.
However, supplementation for only 6 days rather than 12 days, during the oestrous cycle may
prove sufficient to sustain the nutritional influence on ovulation rate and this needs to be
tested because it would reduce costs.
In conclusion, we have provided evidence that a short-term nutritional supplement, perhaps
through its effects on ovarian follicular activity can improve the ovulation rate of ewes
responding to the male effect, but not the incidence of short cycles. The effect on ovulation
rate was not significant with supplementation before male introduction, but this needs to be
re-tested under better experimental conditions. The outcome was favourable when the
supplement was supplied during the male-induced oestrous cycle.
38
CHAPTER 5
5.1 Introduction
Seasonal breeding patterns are caused primarily by the effects of photoperiod on the
pulsatility of GnRH and thus gonadotrophin secretion (Gallegos-Sánchez et al., 1998; Martin,
1984). When the day length is decreasing during the autumn, the inhibition of GnRH
secretion by oestradiol is weak allowing the ewe to experience oestrous cycles and when day
length is increasing in spring and summer, oestradiol gains a strong ability to inhibit the
hypothalamo-hypophyseal system and thus inhibit GnRH secretion, preventing ovulation and
oestrus (Gallegos-Sánchez et al., 1998; Bittman et al., 1985; Martin et al., 1983).
However, the ‘commercial breeding season’ in Western Australia, when farmers place rams
with ewes can commence in late spring and continue until late autumn (Knight et al., 1975),
so it is not well coordinated with the photoperiod-driven natural breeding season. This is
feasible because most of the ewes are Merinos, one of the least seasonal of all sheep breeds –
a proportion of the ewes in a flock ovulates continually throughout the anoestrous season
(Pearce and Oldham, 1988a; Wheeler and Land, 1977; Fletcher and Geytenbeek, 1970). This
phenomenon of ‘shallow anoestrus’ allows us to use the ‘ram effect’ (‘teasing’) to induce
fertile mating and thus advance the mating season into late spring and early summer. The
seasonality of the Merino genotype still causes problems because of the depth of anoestrus
can vary within a ewe as well as a flock of ewes, from season to season (Goodman, 1996).
Also, anovulatory ewes that have been successfully ‘teased’ will return to anoestrous within a
cycle or two if they fail to conceive – a phenomenon attributed to the lengthening daylight
hours during late spring and early summer (Oldham and Cognie, 1980).
Using the ‘ram effect’ to manage reproduction in ewes continues to present two problems:
first, the stimulus by the ram often seems insufficient to induce ovulation, so the response of
ewes varies among and within breeds; second, at the level of the ovary, the follicles destined
to ovulate might not be fully developed and, after ovulation, produce corpora lutea that
regress within 6 days, the ‘short cycle’ phenomenon (Martin et al., 1986).
In the Merino genotype, nutrition is as important as photoperiod for reproduction (Martin et
al., 2002). For most of the summer-autumn commercial breeding season in Western
Australia, feed supply is poor in quality because pasture growth is driven by winter rains
39
(Martin 2014). For this reason, nutritional supplementation (or ‘flushing’) is used at the
beginning of the mating period to boost the energy balance of the ewe and improve
reproductive performance (Scaramuzzi and Martin, 2008). Nutritional supplementation
initiates metabolic processes that increase circulating concentrations of leptin and insulin,
allowing supply of more glucose to the follicles (Scaramuzzi et al., 2006; Leury et al., 1990;
Teleni et al., 1989a; Teleni et al., 1989b). Glucose is the major source of energy for the ovary
and the presence of glucose transporters increases the availability of glucose and other
metabolic hormones for follicle growth (Muñoz-Gutiérrez et al., 2005; Muñoz-Gutiérrez et
al., 2004; Williams et al., 2001). Insulin stimulates the proliferation of granulosa and theca
cells while leptin acts on both the hypothalamo-pituitary axis and the ovary (Muñoz-
Gutiérrez et al., 2005). In females that are already ovulating, short-term nutritional
supplementation does not seem to affect the concentrations of follicle-stimulating hormone
(FSH) and luteinizing hormone (LH) (Downing et al., 1995b); however, the nutritional
signals act directly via the ovarian mechanism (Scaramuzzi and Martin, 2008; Viñoles et al.,
2005) to make follicles more responsive to FSH, allowing extra follicle development.
It is therefore possible that, through actions at ovarian level, nutritional supplementation
might improve the responses to the ‘ram effect’. We tested two hypotheses:
i) That nutritional supplementation will increase the proportion of a flock of ewes that
ovulate in response to teasing in the non-breeding season;
ii) That nutritional supplementation will reduce the proportion of ewes experiencing the
short cycle following ovulation induced by the ‘ram effect’.
5.2 Methods and Materials
The experiment was conducted at Allandale Farm from early December until mid-January
using 112 Merino ewes, all which had lambed at least lambed once, but not in the preceding
lambing season. Prior to the commencement of the experiment (Day -3), laparoscopy was
used to detect cyclic ewes (those with corpora lutea) and acyclic ewes (no corpus luteum
present at the time of laparoscopy). Five minutes before laparoscopy was started, 2 mg
xylazine was injected intramuscularly for partial sedation. At the end of the laparoscopy, 5
mL of benacillin was injected intramuscularly and the wound was sprayed with vetericyn, all
supplied by the Veterinarian who performed the laparoscopy for this experiment.
40
5.3 Experimental design
Cyclic ewes were immediately removed from the experiment. For the duration of the study,
experimental ewes were weighed and condition scored once weekly. The experimental
protocol is shown in Figure 12.
Figure 12: Schematic experimental design.
The Control group (n = 29) was fed hay ad libitum on dry pastures. The experimental
treatments involved nutritional supplements on top of the hay ration.
The treatment group (L-Pre) (n = 29) was fed a supplement before the male effect – lupin
grain (Lupinus angustifolius) at 500g/ewe/day from Day -5 to Day 0.
The Control and L-Pre groups were run separately from Day -5 to Day 0 (the ‘ram effect
period’). On Day 6 of nutritional treatment, Day 0 of the ‘ram effect’, both groups were
placed together in the same paddock and 6 vasectomised rams were introduced (i.e., 1 ram
per 10 ewes). Harnesses with crayons were fitted to the rams for oestrus detection. Ewes
marked within the first 14 days were recorded, but only ewes with distinct crayon marks on
their rumps were part of the data collection and analysis. Crayon colours were changed on
Day 14 and every 7 days thereafter until Day 28. Oestrus observations were also performed
from Days 16 to 26, the expected period of oestrus display by the ewes.
vasectomised rams with
green markers
0 28
marker
colour
20 21
marker
colour
-5
6-day supplement
30
Laparoscopy
14
remove marked ewes,
change marker colour
Laparoscopy
9
Laparoscopy
-2
41
Ovulation rate (ovulations per ewe ovulating) was determined at laparoscopy by counting the
number of corpora lutea on both ovaries of each ewe on Day 10 for first ovulation and on
Day 30 for second ovulation in both groups. Ewes with normal cycle and short cycle were
determined with countback of days from first visible oestrus display.
Statistical analysis
We have based the analysis of treatment effects only on data collected from ewes that had
most probably responded to the ‘male effect’, as evidenced by the occurrence of oestrus
between Days 16 and 26. The number of corpora lutea per ewe ovulating (ovulation rate) was
analysed using Chi-squared tests (χ2) in a 2 x 2 contingency table. Oestrus distributions for
short versus normal cycles were determined using histograms with fits and groups in the
Minitab 14 programme.
5.4 Results
Live weight
Figure 13 shows the live weight data of both groups. The weights were similar for the two
groups throughout the experimental period (P > 0.05).
Figure 13: Average live weight of ewes.
Average (± 0.34) live weight of the Control (unbroken line) and L-Pre (broken line) groups
during the experimental period of 42 days (P > 0.05).
55
60
65
70
1 2 3 4 5 6
Livewight(K
g)
Weeks
42
Ovulation
Table 5.1 shows the percentage of ewes ovulating. In the Control group, the same proportion
16/29 (55%) of ewes exhibited oestrus as ovulated. In contrast, only 10/29 (34%) ewes
ovulated in the L-Pre group. This discrepancy was largely due to 21% of ewes that failed to
show oestrus and they were subsequently excluded from the data.
Ovulation rate
The data for ovulation rate and oestrus for Days 10 and 30 after the ‘male effect’ are also
shown in Table 3. Ovulation rate was significantly higher in L-Pre ewes than in Controls on
Day 10 (P < 0.05), but declined by the second ovulation on Day 30 to be similar to the
Control value.
Table 3: Reproductive variables:
Reproductive variables in Merino ewes induced to ovulate using the ‘ram effect’ during the
anoestrous season. Ovulation data were collected at laparoscopy on Day 10 and Day 30.
Percentages are based on n; different superscripts indicate significant differences (P < 0.05).
Control L-Pre
n 29 29
Ewes ovulating 16 (55%) 10 (34%)
Ovulation Rate1 (Day 10) 1.31a 1.68b
Ewes showing oestrus 16 (55%) 15 (51%)
Ewes showing a short cycle 3 (10%)a 5 (17%)a
Ewes showing a normal cycle 13 (45%)a 10 (34%)b
Ovulation Rate2 (Day 30) 1.40a 1.35a
Ovulatory cycles
Table 5.1 shows the percentage of ewes with normal and short cycles in the two groups. Less
than 50% of the ewes experienced a normal cycle in both groups, but was higher in Control
than in L-Pre (P < 0.05).
Oestrus distribution
Figure 14 shows the number of ewes marked on each day of observation. The ram-induced
ovulation resulted in similar patterns of oestrus for L-Pre and Control (P > 0.05). In both
groups, more ewes were marked between Days 16 and 18 (i.e., a normal cycle length
43
following the ram effect) than between Days 23 and 24 (ewes experiencing a short cycle after
the ram effect followed by a normal cycle). More ewes 16/29 (55%) showed oestrus in the
Control group than in the L-Pre group 15/29 (51%), but the difference was not significant (P
> 0.05).
Figure 14: Oestrus distribution of anovular ewes induced at the ‘male effect’.
Distribution of ram-induced oestrus in animals experiencing a normal cycle (clear bars) or
short cycle (shaded bars). a) Control: an even distribution of oestrus over three days (ewes
experiencing a normal cycle); ewes which experienced the short cycle showed oestrus on
Days 23 and 24. b) L-Pre: an apparently skewed curve in favour of oestrus being displayed
on Day 16 after male exposure; all ewes that experienced the short cycle showed oestrus on
Day 23.
Nu
mb
er o
f ew
es m
ark
ed
by r
am
s
44
5.5 Discussion
The nutritional supplementation did not affect the percentage of ewes ovulating so the first
hypthesis was rejected because the treatment group showed poor ovulation while 38% of the
ewes did not ovulate at all. There was a low response to the ‘ram effect’ in the Control group,
effectively providing the perfect scenario for testing the hypothesis because, if all the Control
ewes had ovulated, there would be no room for improvement with the lupin supplement in the
L-Pre group. This observation confirms other studies with ewes in temperate regions showing
that nutritional supplementation did not respond to the ‘ram effect’, although it seems likely
that increasing the body condition score (BSC) could improve the outcome (Scaramuzzi,
2013, Johson, 2011, Vinoles, 2002). Consequently, a longer period of nutritional
supplementation leading to a high BSC could be a valuable management option (Johnson,
2011).
We used laparoscopy to detect ewes that ovulated by direct visualisation of corpora lutea.
The alternative method of ultrasound scanning might present a better understanding of the
ovarian follicle development, even if it provides less predictive value for follicles < 4 mm in
diameter (Viñoles et al., 2004). On the other hand, our use of lapraroscopy strengthens our
observation of ewes that did not ovulate (Johnson, 2011; Pearce and Oldham, 1988a; Oldham
and Lindsay, 1980; Wheeler and Land, 1977; Fletcher and Geytenbeek, 1970).
We expected nutritional supplementation to improve the responsiveness of ovarian follicels at
the ram stimulus. Our understanding of how small follicles in ewes respond to nutritional
supplementation is still poor and the current information is not consistent. For example,
follicle sizes between 2 and 6 mm in diameter have been resported to escape atresia following
nutritional supplementation, mainly due to changing levels of glucose and metabolic
hormones in cyclic ewes (Ying et al., 2011; Viñoles et al., 2005; Muñoz-Gutiérrez et
al.,2002). In contrast, Rhind and McNeilly (1998) found that nutritional supplementation had
no effect on follicles > 2.5 mm in diameter, but increased the number of follicles 1.0–2.5 mm
in diameter in both follicular and luteal phases.
A small proportion of ewes responding to the ram effect might be due to poor exposure to the
ram stimulus – full physical contact appears to improve the efficacy in ram-induced ovulation
compared to expossure to odour or visual cues (Abecia et al., 2002; Pearce and Oldham,
1988b). It is feasible that tactile cues play a major role and the social interaction among ewes,
as well as between ewes and rams could be important for enhancing the response of ewes to
45
the ‘ram effect’ (Zarco et al., 1995) and in goats (Alvarez et al., 2007). On the other hand,
other reports showed that direct contact with rams was not necessary and have suggested that
the smell and sound are sufficient to induce ovulation (Watson and Radford, 1960). Previous
studies are often very difficult to interpret with confidence because, in the descriptions of
methodology, little is often said about the novelty of the rams used to stimulate the ewes, a
factor we now know to be critical for the ram effect (Jorre De St Jorre, Hawken, &
Martin, 2014).
In the present experiment, short-term nutritional supplementation increased ovulation rate at
the first ovulation, as observed on Day 10. Ovulation rate subsequently returned to control
values after the withdrawal of the nutritional supplementation. Together with the results of
the previous experiment, this observation shows the importance of nutritional
supplementation during the immediate preovulatory period. Glucose and metabolic hormones
at their peak concentrations seem to be major factors in the determination of ovulation rate
(Viñoles et al., 2005; Downing and Scaramuzzi, 1997; Downing et al., 1995a). Nutritional
supplementation lengthens the lifespan of the last non-ovulatory follicle and apparently
prevents the initiation of another follicular wave (Viñoles et al., 2005). In addition, more
follicles of larger size develop when ewes are fed a lupin supplement during the luteal phase
of the oestrus cycle (Somchit et al., 2007; Viñoles et al., 2005). The feeding regime we used
in Chapter 4 supports these findings and the 33% decrease in multiple ovulations in the L-Pre
group at the second ovulaiton suggests that the withdrawal of the supplement led to fewer
follicles growing to the preovulatory stage. Therefore, nutritional supplementation needs to
continue until after the occurrence of the ovulation that is important for the reproductive
efficiency of the flock.
The increase in ovulation rate at the first ovulation is coherent with an expected stimulation
of follicle development by the lupin supplement, but this outcome was not associated with a
reduction in the proportion of ewes experiencing short cycles. We conclude that luteal failure
is independent of the level of nutrition. The occurrence of the short cycle is likely due to
issues at molecular level during luteinization between Day 3 and Day 7 after the ‘ram effect’
(Brown et al., 2014). The uterus also seems to play a role in luteal failure because
hysterectomized ewes showed no evidence of short luteal phase after ram exposure (Lassoued
et al., 1997; Chemineau et al., 1993). This problem is critical because it is preventing farmers
from maximising the value of ram-induced ovulation in flock management (Martin, 2014).
46
In conclusion, a short-term nutritional supplement such as lupin grain can be used to improve
ovulation rate following the ‘ram effect, but has no benefit for number of ewes ovulating or
the occurrence of short cycles. It would be worthwhile to test longer periods of
supplementation because both the proportion of the flock that ovulates and the proportion of
ovulating ewes that go on to show normal cycles are major limitations in the use of the ram
effect for ‘clean, green and ethical’ flock management.
47
CHAPTER 6
6.1 General Discussion
We use the ‘male effect’ as a management tool to induce ovulation in ewes without resorting
to hormone treatments. The general hypothesis of this thesis was that the response of female
Merino sheep to the ‘male effect’ and their subsequent reproductive performance would be
improved by a short-term nutritional supplement. The experimental findings, in combination
with the literature, offer broad support for the hypothesis. However, it is clear that, to
maximise ewe fertility, short-term nutritional supplementation should be fed twice, once
before the introduction of rams and again before the second ovulation, during the ram-
induced oestrous cycle.
We expected short-term nutritional supplementation to increase the number of ewes that
ovulate in response to the ‘male effect’, but the results did not support this hypothesis. The
experiment might have been compromised because it was done at the start of the mating
season and many ewes were beginning to ovulate spontaneously. However, even in the ewes
that were still anovulatory, nutritional supplementation did not improve the outcome. Our
knowledge on how nutritional supplementation affects ovarian dynamics is not complete,
especially for anovulatory sheep, but it is still likely that nutritional stimulus increased
follicular activity (Viñoles et al., 2005; Somchit et al., 2007). The most logical interpretation
is that the outcome of the male effect, in terms of whether the female ovulates or not, is not
determined at the level of the ovary, but most probably at the level of the brain-pituitary
systems that control gonadotrophin secretion.
After ovulation is induced by the male effect, the next question is whether the female has a
single or multiple ovulations. In this case, the importance of nutrition is very well
documented for ovulatory Merino sheep. We can now extend this to ovulation induced in
anovulatory ewes because, in both experiments, ovulation rate at the ram-induced ovulation
was increased by nutritional supplementation. We can therefore conclude that an increase in
energy supply enhances ovarian follicular development so more follicles can ovulate in
response to the prevoulatory surge, whether the surge is induced by the male effect or during
the normal processes of spontaneous ovulation (Scaramuzzi et al., 1993, 2006, 2011).
48
The overall outcome was an improvement of reproductive performance in ewes by nutritional
supplementation at the ram-induced ovulation. Moreover, a nutritional stimulus after the ram-
induced ovuation, in the period leading the the second ovulation, also significantly improves
ovulation rate, as demonstrated in Chapter 4. These findings suggest that, rather than aiming
for precise timing, farmers would be better off ensuring that the duration of the feeding is
sufficient to cover the second cycle.
Farmers can be confident in this plan because when nutritional supplementation is maintained
in the period between October and March, ovulation rate is also maintained and not affected
by season, whereas the response to single, short-term supplements diminishes in December
before recovering in March (Gherardi & Lindsay, 1982). As shown in Chapter 4, a second
supplement near the time of luteolysis after the first ram-induced ovulation seems to avoid
the decrease of ovulation rate experienced at the second ovulation in Chapter 5 where only a
single nutritional supplementation was given before the first ovulation induced by the male
effect.
One other limitation of the present experiments was the need to run the ewes in treatment
groups, with the ewes trail-fed in a paddock. This design is difficult to avoid with field
studies and makes it difficult to ensure that each ewe has received an appropriate amount of
the lupin grain, with perhaps the most dominant ewes consuming more than the lower
members in the hierarchy. On the farm, farmers dealing with large numbers of production
animals cannot feed them individually so, although compromised, the experiment reflected
reality.
The hypothesis that short-term nutritional supplementation would prevent or reduce the
proportion of ewes experiencing a short cycle after the ram-induced ovulation was rejected.
As stated above, we have little knowledge on how nutritional supplementation affects ovarian
dynamics in anovulatory sheep, but we do know that, in the present experiments, these
supplements allow an increase in ovualtion rate so there are clear benefits for follicular
function. We therefore feel confident that the failure of the ram-induced corpus luteum is not
caused by the ovulation of follicles that are poorly developed because of nutrition-dependent
factors. In other words, the same nutritional signal that improves ovulation rate at the ram-
induced ovulation does not affect the destiny of the the ram-induced corpora lutea. Other
factors must therefore be the dominant cause of short cycles, such as premature luteolytic
49
signals from uterus (Southee et al., 1988; Chemineau et al., 2006) or poor activation of genes
that are critical for the development and function of the corpus luteum (Brown et al., 2014).
We seem to be gaining an understanding of the cause of short cycles following ram-induced
ovulation, but need much more progress towards unpacking the phenomenon if we are to
provide a solution relevant to farmers.
Future areas for research
We need more research to help us overcome the shortcomings of the ‘ram effect’, such as the
failure of some ewes to respond by ovulating and the short cycle phenomenon. The short
cycle is particularly important for practical applications on farm because it ruins an otherwise
excellent level of synchrony of ovulation and lambing in the ewe flock. The work of Brown
et al., 2014 clearly shows the value of exploring the molecular processes that underpin the
phenomenon. A solution would allow us to gain far more value from the ram effect in the
implementation of ‘clean, green and ethical’ management of sheep production.
Namibian perspective
The repeatability of the present experiments under Namibian conditions remains to be tested.
However, it seems likely that farmers in Namibia could use the male effect to adjust the
mating season to ensure that the lambing coincides with the best grazing to support lactation.
Indeed, Namibian farmers need to incorporate a ‘smart feeding regime’ that takes into
consideration the long dry spells the country experiences each year. There have been some
attempts to do this, with breeding programmes commencing in July so that lambing and
lactation coincide with the rainy period from early December to late March, but the entire
reproductive process has not been followed to provide quantitative information.
To date, most livestock research in Namibia has focussed on the breeding of hardy, well-
adapted animals. We can build on this as well as our general understanding of the value of
nutrititional supplementation for improving reproductive performance, but we need to design
locally-relevant projects that will allow the adoption of the ‘clean, green and ethical’
concept. We also need to investigate the extent of reproductive wastage under arid conditions
and identify sources of wastage that can be mitigated. Finally, we need a full understanding
of reproductive performance across the broad spectrum of livestock production systems that
exist in Namibia.
50
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