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Romanian Biotechnological Letters Vol. 15, No.3, 2010, Supplement Copyright © 2010 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

3

The retrospective results of the research developments regarding reproduction biotechnologies in sheep and goat in romania

Received for publication, March 2, 2010

Accepted, May 3, 2010

STELA ZAMFIRESCU Faculty of Natural and Agricultural Sciences, “Ovidius” University, 900527, Constanta, Romania Tel/fax 0241620063, [email protected]

Abstract

In Romania, the assisted reproduction technologies in sheep and goats (ART) have been used since 1978 with the purpose of maximizing the productive potential in small ruminants. The first generations of reproduction biotechnologies, the synchronic induction of estrus with artificial insemination in succession, have been successfully applied in Romania since the 1960s-1970s by OTEL & PARASCHIVESCU [3]. After 1978, the technologies for collection, dilution, preservation and use of semen were doubled by the more intense use of second generation biotechnologies, semen freezing and embryo transfer. In parallel with the technological and scientific development of the reproduction biotechnologies, after 1994 the following began to be used: the biotechnologies associated to the embryo transfer such as in vitro fertilization and embryonic bisection (ZAMFIRESCU, [17, 18, 33, 37, 41]. The ART advantages are unanimously recognized (sanitary and veterinary, zootechnical, scientific and economical), but their application in practice is still limited because of the financial obstacles but also because of the lack of super-qualified labor. The researches done in Romania in the field of artificial insemination in sheep and goats have been extremely numerous such as: the optimization of dilution media, the optimal glycerol level which ensures the protection of the sperm cells, the freezing of the buck semen with or without seminal plasma, the establishment of the best methods and type of freezing, the optimization of the insemination methods for cryopreserved semen and the quality assessment of frozen-thawed semen by different methods (S. ZAMFIRESCU, [31, 34]. Our results in the freezing of the ram and buck semen are very good, comparable to those obtained at international level (B. LEBOEUF, [42]. We founded a cryobank for ram and buck semen (over 10 000 straws/stock), which served to deposit semen from active populations and from population in genetic preservation. Over the past 15 years, the results in the sheep and goat embryo transfer were from a number of 450 sheep, highlighting an ovulation rate of 3-12 after treatments with eCG and 5-16 after treatments of superovulation with FSHo and FSHp. The number of viable embryos was between 3-8, according to treatment and especially according to the individual variability of the donors. The pregnancy rates were between 45-65 % in sheep and 65-70% in goats. The embryo survival was between 35-32 % (S. ZAMFIRESCU, [4]. In vitro fertilization is a very complex biotechnology studied on local sheep and goats in Romania. The oocytes are collected from ovaries of slaughtered females, are matured, fertilized in vitro and cultured for 6–7 days when the fertilized oocyte develops into preimplantation morula or blastocyst. In Romania, the coefficient of transformation from complex cumulus-oocytes in vitro maturation and fertilization is 73% (maturation) 47% (cleavage in 2-4 cells) 26 % (preimplantation morulae) and 9% (blastocysts). This transformation of complex cummulus-oocytes is very unsatisfactory because it is conditioned by many factors.

Keywords: biotechnologies, semen, embryo, sheep, goat Introduction

In Romania, the assisted reproduction technologies in sheep and goats (ART) have been used since 1978 with the purpose of maximizing the reproductive potential in small ruminants (ZAMFIRESCU, [12]; IONESCU, [2]. In the local sheep and goats, there was a greater need to improve the production and reproduction performances, as the accent was on the exploitation for milk and meat. The first generations of reproduction biotechnologies, the


Romanian Biotechnological Letters, Vol. 15, No. 3, Supplement (2010) 4

synchronic induction of estrus with artificial insemination in succession, have been successfully applied in Romania since the 1960s-1970s by PARASCHIVESCU & OTEL [3], ZAMFIRESCU & SONEA, [4]. After 1978, the technologies for collection, dilution, preservation and use of semen were doubled by the more intense use of second generation biotechnologies, semen freezing and embryo transfer. In parallel with the technological and scientific development of the reproduction biotechnologies, after 1994 the following began to be used: the biotechnologies associated to the embryo transfer such as in vitro fertilization and embryonic bisection (ZAMFIRESCU & al, [4,5,6]. The ART advantages are unanimously recognized (sanitary and veterinary, zootechnical, scientific and economical), but their application in practice is still limited because of the financial obstacles but also because of the lack of super-qualified labor (AMOAH & GELAYE, [7].

At international level, the success ART has had in all countries with developed zootechny, demonstrated that, if the technologies are respected, the reproduction in sheep and goats is maximized and high rates of fecundity are obtained after artificial insemination with frozen semen (MAPLETOFT, [8]; LEBOEUF & al. [9]; BARIL, [10]. Higher conception rates (50-75%) are also obtained by the use of frozen and fresh embryos (BARIL, 10], as well as higher conversion rates of the oocytes in the embryos produced in vitro (COGNIE & al, [11].

Ten years have passed from the birth of the first cloned animal – Dolly the sheep (WILMUTH & al, [12]. The recent techniques in ART embryology facilitated the development of the transfer of desired genes or even of the entire genome of certain valuable animals. Also, the rapid development of the techniques for the manipulation of gametes, especially of embryos, made possible the genome screening and the highlighting of certain genetic defects or the quantitative increase of the genetic traits by the use of molecular markers (GORDON, [13]; CAMPBELL & al, [14]; COLMAN & al, [15].

The ART techniques include the induction and synchronization of estrus with application in succession of the artificial insemination with semen preserved in different manners, the synchronization of parturition, the production of multiple ovulations and embryo transfer, the freezing of gametes, in vitro fertilization, the production of embryos in vivo or in vitro, the bisection of embryos, cloning, the production of transgenic animals and the immunological diagnosis of pre-implantation.

This article describes the main achievements in the field of ART in sheep and goats: the induction and synchronization of estrus, artificial insemination with frozen semen, the production of embryos in vivo and embryo transfer, semen and gamete freezing, in vitro fertilization and embryonic bisection.

1. Induction and synchronization of estrus in sheep and goats

Between 1960-1990, large numbers of sheep and goats were used for the induction and synchronization of estrus by the use of injections of synthetic progesterone (4 injections every 3 days for 12 days). On day 12, eCG (PMSG) is administered in doses of 500-700UI. The results of this conception recipe have always been constant with values between 40-65% depending on season and breed (ZAMFIRESCU & IONESCU, [16]; ZAMFIRESCU, [1], [17]. At The Research and Development Institute for Sheep and Goat Breeding- Palas Constanta, the Laboratory for Reproduction and Biotechnologies, we experimented different ways to administer progesterone with the purpose of reducing the number of interventions per animal, progesterone preparations for oral administration or collagen implants for vaginal insertion (Progestcol G10), which had the advantage of slow resorption without the need to extract sponges. The results were relevant in terms of estrus induction and enhancing fertility after artificial insemination (Table 1). The schemes for induction and synchronization of estrus were applied on sheep and goats in estrus during normal reproduction season and

STELA ZAMFIRESCU


counter-season, using different types of progesterone and different ways of administration (ZAMFIRESCU, 18], AMIRIDIS & al, 19]; AMOAH & GELAYE, [7]. During the same period, at international level, there was a successful use of vaginal sponges impregnated with synthetic derivates of progesterone such as fluorogestone acetate (45 mg FGA) or medroxyprogesteron acetate (60 mg MPA). The use of progesterone in combination with FSH or eCG in the schemes of induction and synchronization of estrus and superovulation led to different results, sometimes inconstant. The results were similar to those obtained with Chronogest in France and others countries (WHITLEY & al, [20]; WILDEUS, [21]; DONALDSON, [22]; DRIANCOURT, [23]; BOSCOS & al, [24]).

Table 1. The efficiency of treatment with Progestcol G10 in Merinos of Palas sheep during seasonal non-estrus (March 1989 and April 1990).

Treatment period

Group Treated females(n)

Estrus* n/%

Females calving(n)

F(%) P(%) TE(%)

April `89 Treated 120 83/69.16 61 73.49 157.37 50.83 Witness 120 23/19.16 19 82.90 115.79 15.83 March `90 Treated 226 169/74.77 143 63.27 143.55 63.27 Witness 85 20/23.52 16 18.82 125 18.82

*in a period of 12 days; F: fertility; P: prolificacy; TE: treatment efficiency 2. The production of multiple ovulation for in vivo embryos and embryo transfer in

sheep and goat The induction and synchronization of estrus in sheep and goats as well as the

treatments for superovulation permit the improvement of reproduction efficiency and the use at maximum potential (DRIANCOURT,[ 23]; BOSCOS & al, [24]; D’ALESSANDRO & al, [25]; LOPEZ, [26]; RIESENBERG, [27]. These technologies permit the easy application of complementary techniques for reproductive management, including artificial insemination and embryo transfer so that that genetic material is easier to obtain and manipulate (DRIANCOURT & FRY, [28].

The superovulation treatments were discovered approximately 50 years ago and implemented in research and production in sheep and goats (DRIANCOURT [23, 28]. The FSH-based treatments (the follicle-stimulant hormone) or eCG-based ones (the serum gonadotropin from pregnant mare) cause the development of multiple follicles followed by multiple ovulations.

In the practice of our laboratory, the programs of synchronic induction of estrus and multiple ovulation used for the first period the progesterone treatments (synthetic progesterone) or Chronogest in association with eCG or pituitary gonadotropins (as commercial preparation, FSHp,FSH o, LH, Stimufol). The luteolisis of the residual corpora lutea was induced by the administration of PgF 2α in variable doses, depending on the applied treatment (Cloprostenol, Flavoliz, Revoliz).

These treatments were followed by natural mating or artificial insemination, monitoring the females for pregnancy in receiving ones. The superovulation treatments in sheep and goats were evaluated by laparoscopy before the surgical collection of embryos or directly at the moment of collection. The collection and transfer of embryos was made by the surgical method, from donor sheep and goats anesthetized according to the usual practices.



We mention that the usual washing PBS media with BSA4% were used 95% of the time, using the washing on days 5-7, by means of the TomCat and Folley probes on the direction of the superior third of the uterus horns. The methods for the appreciation of embryo quality were done based on the morphological analysis. The collection, analysis, preservation and transfer of embryos respect the conditions imposed by the IETS norms (IETS, [29].

Over the past 15 years, the results in the sheep and goat embryo transfer were from a number of 450 sheep, highlighting an ovulation rate of 3-12 after treatments with eCG and 5-16 after treatments of superovulation with FSHo and FSHp.

The number of viable embryos was between 3-8, according to treatment and especially according to the individual variability of the donors. The pregnancy rates were between 45-65 % in sheep and 65-70% in goats. The embryo survival was between 35-32 % (ZAMFIRESCU, [30, 31].

The general results are confirmed by the experiments done on goats in 2002 (Table 2) (ZAMFIRESCU, [32]. The highest ovulation rate (14) is obtained after treatments with FSH-p and 13,7 after treatments with PMSG and Neutra –PMSG. The transferable embryos were between 9-12. Noticeable here is the percentage of females with positive reaction of only 59% of the total, compared to 13-14% after treatment with FSHp and eCG-NeutraPMSG.

The results in tables 3 and 4 show the results of the conception rate after transfer in receiving sheep and goats. These rates were influenced by the treatment method. Thus, a rate of 62.5% is obtained in goats after superovulations with eCG and rates of 75-83% are obtained after treatments with Neutra PMSG and FSH p. The embryo survival was acceptable, of 33.33-58.33%.

In sheep, the conception rate is variable according to breed, semen origin and the preservation period of the frozen embryos. Thus, by the transplant of frozen embryos, the conception rate was between 30-48.18%, while the embryo survival was 43-61.94% (ZAMFIRESCU, [33,34,35]. Table 2. Embryo production in local Carpathian goats after different treatments (2002)

Transferable embryos

Group-Treatment

Treated female(n)

Female With reaction (n / %)

Ovulation rate

Nonovulated follicles (n) Total

n / %

E/ donors (%)

I- PMSG 12 7 / 59 9.0 6.8 33(68) 3.66

II- PMSG +NeutraPMSG 10 7 / 70 13.7 2 79(93) 11.28

III- FSH-p 11 9 / 81 14.0 0.6 85(89) 9.14

STELA ZAMFIRESCU


Table 3. Kids obtained after freezing goat embryos

Group-Tretament Receptors (n)

ET* (n)

Receptor (n / %)

Born kids (n)

Embryo survival (%)

I- PMSG 8 18 5 / 62.50 98 44.44

II-PMSG+ NeutraPMSG 8 16 6 / 75.00 9 11 68.75

III- FSH-p 6 13 5 / 83.33 7 10 76.92 *ET: transferred embryos

Table 4. Results concerning pregnancy rates obtained after the transfer of freezing-thawing ovine and caprine embryos

Years Embryos (n)

Breeds Recipients (n) New Borns(n)

Embryo survival (%)

1996 16 *Lacaune 6 /37.5 7 43.75

1997 25 *Ille de France 9/36 12 48.00

1998 12 Saanen 4 /30 7 58.33

1998 6 *Angora 3 /50 2 33.33

1998 13 **Local goats 6 /46.15 7 53.85

1999 21 *Palas Merinos ewes 9 /42.85 13 61.94

2000 27 *Palas Merinos ewes 13 /48.18 15 55.55

*embryos imported from France in September 1993;**Embryos freezing in LRB-Palas Constanta

3. In vitro fertilization (FIV) FIV is the very complex biotechnology but it is a very good alternative for superovulation induction. The oocytes are collected from ovaries of slaughtered females, are matured, fertilized in vitro and cultured 6–7 days when the fertilized oocyte develop into preimplantational morula or blastocyst. In our laboratory, the coefficient of transformation from complex cumulus-oocytes in vitro maturation and fertilization is 73% (maturation) 47% (cleavage in 2-4 cells) 26 % (preimplantational morulae) and 9% (blastocysts).

This transformation of complex cummulus-oocytes is very unsatisfactory because it is conditioned by many factors. Apart from AI and ET, the other associated biotechnologies in goat reproduction are not very efficient because they are very expensive (TURCU & ZAMFIRESCU, [36]; ZAMFIRESCU & NADOLU, [37, 38].



Fig 1. Sheep oocyte, morulae of 2 and 4 cells, and bastocyst ( Zamfirescu,1997) 4. Artificial Insemination in goats and ewes

The researches done in Romania in the field of artificial insemination in sheep and goats have been extremely numerous (ZAMFIRESCU & NADOLU, [37,38]; RITAR & SALAMON, [39]. Their objective was the optimization of dilution media, the optimal glycerin level which ensures the protection of the sperm cells, the freezing of the buck semen with or without seminal plasma, the establishment of the best methods and type of freezing, the optimization of the insemination methods for cryopreserved semen and the quality assessment of frozen-thawed semen by different methods (ZAMFIRESCU, [18, 40, 41]. Our results in the freezing of the ram and buck semen are very good, comparable to those obtained at international level (LEBOEUF & al, [42]; SALAMON & MAXWEL, [43].They materialized by the establishment of a cryo-bank of semen (over 10 000 straws/stock), which served to deposit semen from active populations and from population in genetic preservation.

I mention that out Laboratory has offered, at the demand of understandings between the Agriculture Ministers from Sofia and Bucharest, over 1000 doses of frozen semen (Australian Merinos). The semen was transported to the Sumen Station, Bulgaria and it offered in exchange buffalo semen (1985;1987).

The semen was frozen in vials (1978-1990), in medium vials (1990-1994) and currently, it is frozen only in fine straws. Numerous researches were done for the experimentation of certain dilution media, among which, the best results are obtained with saline extenders (Tris and Citrat), those based on milk (media imported from France, such as Regillait and Gaillard), more recent ones (Biladyl, Triladyl) and media from natural juices of fruits and vegetables. The motility is evaluated on all the phases of the technological flux of semen freezing according to a technology of the LRB. The results are materialized by motilities after thawing between 35-60%. The conception rates are extremely variable, depending on many factors such as semen quality, optimal insemination time, insemination type, female factors and the inseminator’s routine (RITAR & SALAMON, [39]. The quality of the seminal material is established by the determination of the following sperm and cryobiological indexes: volume, concentration, motility of diluted semen (M2%), motility

STELA ZAMFIRESCU


after thawing 24 hours after thawing (M6%), sperm cell survival (SS%), cryobiological adaptability of the ejaculates (CP%) and cryobiological loss (CL%).

A few of the results obtained in the Laboratory for Reproduction and Biotechnologies are presented in Tables 5, 6 and 7. The final results prove that the results obtained in our laboratory are very variable according to breed, media used, glycerin level and semen quality after thawing.

Table 5. The cryobiologic index of Carpatina and Saanen X Carpatina buck semen freezing in milk extenders (2001)

Breed Ej (n)

Fresh Motility %

Motility after thawing (%)

Adaptability index %

Survival index %

Cryo Reject %

Carpathian/2000 29 95±2.67 38.77±6.37 77.77 43.07±6.51 22.22

Carpathian .2001 68 90±3.77 49.28±8.95 87.5 51.87±10.49 12.5

White of Banat/2000 48 95±2.58 43.76±4.68 75 47.25±5.39 25

White of Banat/2001 59 90±2.58 39.97±9.74 66.66 44.41±11.44 33.33

Crossbreed(Saanen XCarpathian) 2000 48 90 37.39±0.17 62.5 41.54±0.19 37.5

Crossbreed (Saanen X Carpathian) 2001

47 90±2.58 39.28±8.05 85.71 41.34±9.48 14.28

Crossbreed (Saanen X Carpathian)2002

59 92.5±2.73 49.69±6.81 66.66 53.575±5.77 33.33

The determination of semen quality by electron microscopic exam demonstrated the

deterioration of the head of the sperm cell (40-60%) with the partial or total destruction of the plasmatic membrane at the head level and less at the flagellum level (Fig. 3).The pregnancy rates were variable, between 20 and 75% (SALAMON & MAXWEL, [43]; ZAMFIRESCU & al, [44,45].

Table 6. The cryobiologic index after freezing buck semen in different extenders (2004)

Citrat Triladyl Biladyl Parameters

n X ± SX n X ± SX n X ± SX

Glycerin 5% 28 46.8±0.13 30 48.7±0.15 38 49.4±0.12

M6 % 28 46.43±2.27 30 43±3.14 38 43.68±1.96

ME/N

% 28 52.02±2.07 30 45.73±2.33 38 48.52±1.69

IS % 28 63.37±2.28 30 51.86±2.49 38 54.04±1.71



Table 7. Semen and cryobiological indexes of ram sperm cells frozen in TRIS, with different glycerin levels

Tris+glicerina(%) (n) Motility fresh semen (%) X± sx CV%

Motility after thawing(%) X± sx CV%

Survival index(%)

3 12 85±8.13 3.50 43.08±5.18 11.06 47.05 4 12 85±8.53 3.13 45.08±6.33 15.79 53.03 7 12 87±5.87 4.05 26.33±15.36 15.36 41.75

a)Ram fresh semen b)Ram frozen semen

c)Buck fresh semen d)Buck frozen semen

Fig 2. The ultrastructure of ram and buck sperm cells in the freezing - thawing dynamics (Zamfirescu S, 2004) Conclusions The frozen semen of ram and buck must have a minimal post-thawing motility of 35% in order to be deposited in the frozen semen bank. It must be inseminated in sheep and goats only in the cervix, under vaginoscopic control, which should lead to a minimal conception rate of 40%, accepted by breeders and farmers. Acknowledgments This research was realized within the National Research Program CEEX no. 10522; 3-389 and RELANSIN 1732, subsidized by the Ministry for Education and Research, Bucharest. The author thanks assistant professor Olivia Chirobocea for the translation of the article into English.

STELA ZAMFIRESCU


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