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Fertility assessment -spermatozoa Dr. S. Selvaraju, Scientist (SS), Reproductive Physiology Lab, Animal Physiology Division, National Institute of Animal Nutrition and Physiology, Bangalore-560 030 NATIONAL INSTITUTE OF ANIMAL NUTRITION AND PHYSIOLOGY (Indian Council of Agricultural Research)

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Fertility assessment -spermatozoa

Dr. S. Selvaraju, Scientist (SS),

Reproductive Physiology Lab,Animal Physiology Division,

National Institute of Animal Nutrition and Physiology, Bangalore-560 030

NATIONAL INSTITUTE OF ANIMAL NUTRITION AND PHYSIOLOGY

(Indian Council of Agricultural Research)

Importance of semen evaluation Fertility and sub/infertility Seminal plasma and its role in fertility Seminal plasma proteins and capacitation Fertility proteins (factors) in seminal plasma Anti-fertility proteins (factors) in seminal plasma Enhancing fertility of low fertile bulls with highly

fertile bull seminal plasma

The key to a cattle breeding program -reproductive management

Reproductive management — heat detection, artificial insemination (AI), pregnancy detection, etc

Artificial Insemination (AI) -genetic gain and reproductive progress in a shorter period of time.

The only competition to AI is natural service -Many bulls have a low libido, low fertility, and

can carry many venereal diseases

Low fertility in AI

Various factors influences fertility

Selection of sire -vital part of AI

Accurate fertility prediction in male is essential for high reproductive efficiency

Standard procedures of semen evaluation

Physical characters- colour, odour, viscosity, pH, etc

Physiologic integrity of sperm-sperm survival

Membrane integrity testsTransportFertilizing ability

Standard procedures of semen analysis are relatively poor indicators of in vivo fertility

Fertile, Sub-fertile and Infertile maleswith Abnormal semen parameters

Poor sperm motility

Abnormal spermatozoa

Poor freezers

with normal semen parameters

Factors unknown

Fertility regulating substances

No single test has been developed in vitro to predict fertility/ conception rate in vivo accurately

No single test is sufficient to predict fertility

Fertility predicting test (s) – array of tests

simple functional tests to molecular tests

successful fertilization and embryonic development

Fertility prediction, the search is on and on….

Sub-fertility- serious concern in dairy industry(In human, 40% due to male, M:F= 1:1)

Nutrient related metabolites such as insulin, GH, and IGFs - support the gonadotrophin-dependent events

Based on IGF-I levels fertile bulls can be selected for breeding/AI (Yilmaz et al., 1999)

Functional and molecular tests / markers in semen influencing fertility

 Predicting and enhancing semen fertility

Sperm Function tests –Routine semen analysisMass activity

Concentration

3. Hemocytometer

2. Photometer

3. CASA

Progressive Forward Motility and abnormalities-Subjective

Progressive Forward Motility and abnormalities-CASA

VSL is correlated to fertility

Criteria for selection: Mass activity = 3+PFM = >60%concentration = >500million cells /mlAbnormalities = <20% ( total)

Parameters of fertility prediction tests in semenI. Function tests:

(a) Progressive forward motility(b) Plasmalemma integrity(c) Acrosome integrity(d) Functional membrane integrity (e) Sperm nuclear morphology(f) Sperm nuclear decondensation (g) DNA fragmentation (h) Mitochondrial membrane potential(j) Hypo-osmotic swelling and acrosome integrity

II. Molecular/Biochemical tests : (a) Assessment of seminal plasma and sperm membrane proteins and minerals(b) Heparin binding proteins in semen

III. In vitro fertilization tests:(a) Sperm-zona pellucida binding assay(b) In vitro sperm penetration assay(b) Cleavage rate

Plasmalemma integrity –vital stainsa

b

c

a: Live; b: Dead; c: Moribund

Plasmalemma Func. Memb. Integrity - HOST

Acrosome integrity

Assessing acrosomal integrity alone not sufficient to assess semen fertility

Comet assay

Most common semen evaluation tests - inconsistent correlation with fertility in cattle and human

(Aitken, 2006).

Intact and functionally active membrane is required for sperm metabolism, capacitation, ova binding and acrosome reaction

(Jeyandran et al., 1984).

The independence of different regions of plasma membrane surrounding the sperm

Subpopulations of spermatozoa have been characterized based on functional membrane status and acrosome integrity in pigs

(Perez-Llano et al., 2003).

Combination of tests

Functional membrane integrity and acrosomal integrity

Protein profiles in semen

Need of the hour for AI industry Selection of sire based on – BSE and standard semen evaluation

Variation in bull fertiltiy-25%- Subfertility?

Fertility evaluation trials?

Fertility evaluation after insemination – expensive and time consuming

Accurate fertility diagnosis in vitro is essential for high reproductive efficiency

Seminal plasma Complex mixture of secretions

Testis

Epididymis

Accessory sex glands

Contains, several polypeptides Some proteins binds to spermatozoa

role in fertilizing ability of sperm

Seminal plasma & fertility with controversiesMammalian sperm acquires fertilizing ability in the epididymis

Cauda epididymal sperm fertility is less than ejaculated sperm

Removal of accessory sex glands esp. seminal vesicles

Detrimental to in vivo fertility

Retarded embryo development

Implantation loss

Addition of seminal vesicle fluid in the cauda epididymal sperm

Increases sperm velocity

Alterations in motility pattern

Proteins, minerals, energy substances, etc., in seminal plasma alter sperm function.

In vivo, seminal plasma does not accompany sperm to the site of fertilization

Decapacitation factors should be removed, modified or masked before acrosomal reaction.

Heparin binding proteins69*55*

(Osteopondin)30*

(FAA)23*

HB Protein profile in seminal plasma and sperm membrane between different fertile bulls

Analysis of HBP concentration may be helpful to assess fertility

BSP Proteins Production - testosterone dependent

Originate from seminal vesicles

Binds to high density lipoprotein

Calmodulin

Heparin

Added to sperm membrane during ejaculation

BSP protein Head Capacitation

Acrosomal reaction

ovum recognition Epididymal semen -post acrosomal region

preference over mid piece regionSperm mid piece – mitochondria Spermatozoa metabolism

Seminal plasma protein and capacitationEjaculation

BSP + Phospholipids on sperm head(Decapacitate spermatozoa - prevents early acrosome reaction)

Female Reproductive tract – HDL

BSP + HDL (lipoprotein)BSP- sequester cholesterol, Phospholipids

(Leaves sperm surface)

Altered permeability of sperm membrane ---Calcium entersActivation of phospholipase A2

Bovine seminal plasma protein

26kDa proteinLipocalin type PGD synthase

Secreted by testis and epididymis

Cauda epididymis Secrets 8% more protein than seminal vesicles

Concentration depends on Collection frequency Present in luminal fluids

3.5% greater in bulls with above average fertility

Lipocalin

55kDa proteinHighly acidic, Phosphorylated glycoproteinIsolated in many speciesFound in Milk, urine-66kDa

Testis, epididymis and sperm

Fertility regulation is by…..8. Affecting sperm physiology9. Influencing events associated with the female

Osteopondin

Osteopontin was responsible for 48 % of the variation in non-return rates of the bulls

Molecular weights of secreted osteopontin vary from 25 to 80 kDa- Many isoforms (14 to 55 kDa)

Heparin Binding proteins on spermHeparin

Secreted in the female RP tract Sperm affinity to heparin-Protein on sperm

Capacitationacrosomal reactionfertility

Male accessory glands secretes HBP

Produced under androgen controlBinds with spermatozoa at ejaculation5 affinity proteinsHBP-B5-Greatest affinity in sperm membrane

Multiple protein. 14 to 30 kDa.

Important –21.5, 24 and 30 kDa HBP

HBP on fertility….Bulls sperm with greater affinity for

heparin have increased fertility

17% more in fertile bulls

HBP on sperm membranes but not in seminal fluid had greater fertility

Fertility Associated Antigen 30 kDa protein

Membrane proteins in freezing

Anti-fertility factors in seminal plasmaDecapacitation factor

Human anti-fertility factor –1(AF-1)

Non dialysable and heat labile

Rabbit acrosome stabilizing factor (ASF)

Bull seminal plasma (SPLN) inhibitors

Bull seminal plasma acrosin inhibitor(BUSI-I)

capacitation and Acrosome reaction

Acrosomal enzymes-corona/zona Penetration

Interfere with fertilization.

Enhancing fertility of low fertile bulls with highly fertile bull seminal plasma

BSP protein (fertility and anti-fertility protein) associated with sperm memb. at the time of ejaculation

Association is quite strong

Washing and mixing of ejaculated sperm with seminal plasma of other bulls may not influence fertility

Mixing Epididymal spermatozoa of low fertile bulls with highly fertile bull’s seminal plasma may enhance fertility

Role of buffalo sperm DNA quality in influencing Role of buffalo sperm DNA quality in influencing fertility and embryo qualityfertility and embryo quality

Role of male in embryo survivalRole of male in embryo survival

AI bulls –Variation in NRR by 20 to 25 %,- routine semen analysis? Subfertile sires-normal semen quality

(Killian et al., 1993; Larson and Miller, 2000)

Fertilization - Contribution of male > 50% in AI

Differences in embryonic mortality between bulls (Hillery et al., 1990; Shi et al., 1990)

Embryos from fertile semen cleaves faster than in/sub fertile

DNA abnormal quality low fertilization rates, impaired pre-implantation development, increased abortion

(Lewis and Aitken, 2005) Sperm nuclear abnormalities -not correlated to other semen parameters,

but positively correlated to fertility (Selvaraju et al., 2007)

Sperm DNA condensation/fragmantation-No studies in buffalo

Sperm DNA qualitySperm DNA quality

Buffalo bull spermatozoa prone to oxidative stress

less antioxidant enzymes

higher lipid peroxidation

More DNA damage as compared to cattle

Abnormal DNA quality Disrupted spermatogenesis

Lipid Peroxidation

External factors heat stress

(Sailer et al, 1997)semen extenders

(Karabinus et al, 1991)genetic (Peris et al, 2004).

DNA distribution / Sperm Nuclear Morphology

Most of the sperm evaluation methods the viability functional status of spermatozoa, influence the fertilizing ability of the gamete.

Vital factors essential for the sperm to produce a healthy and viable offspring

depends on the genetic material content

Such structural or biochemical defects are thought to be associated with chromatin packaging in the sperm nucleus.

Sperm with non-compensable defects -abnormal DNA distribution -improper zygotic, embryonic and or fetal development.

Sperm DNA is uniquely condensed and organized abnormality associated with DNA, chromatin packing, or the sperm nuclear matrix

change in sperm nuclear shape.

The evaluation of sperm nuclear morphology should be useful for assessing male fertility.

Sperm Nuclear Morphology

Causes of improper DNA distribution/ nuclear lesions

DNA distribution/chromatin condensation -independent of conventional sperm parameters like, morphology, count and motility.

Seasonal fluctuation of nuclear lesions( 1-26%)

Stress due to transportation and environmental

chemical (ethylene dibromide, grain fumigant)

Hormonal imbalance

Proportion of spermatozoa double stranded DNA decreases after freezing and thawing

These lesions may not necessarily due to genetic.

Sperm - oocyte binding assay

Zona-free homologous oocyte penetration assay

Zona free oocyte

Disadvantage2. Handling- Many times-Loss of oocytes3. Staining is must4. Block to polyspermy-Mature oocyte

Immature Homologous Oocytes Penetration Test

Sperm-zona penetration

Sperm-zona penetration cont….

IVF

Unfertilized Fertilized: pronucleus

Cleavage: 2-cell

MF

Stages of fertilization

Can the fertilizing capacity of a given semen sample be predicted in vitro?

3. Fertilization depends on several interrelated characteristics4. Semen evaluation is complex and unknown factors influences

fertility5. Still what combination is effective?6. Semen evaluation invitro and as well as sperm-oocyte

binding/penetration/cleavage- useful to predict fertility

Improving Semen fertility

Identity of humoral mediators involved in nutrient mediated reproductive events remains equivocal (Brito et al., 2006)

Glucose, AA or nutrient related metabolites, such as insulin, GH, and the IGF’s and IGFBP’s, operate at the gonadal level and to support the gonadotrophin-dependent events

Based on IGF-I levels fertile bulls can be selected for breeding/AI (Yilmaz et al., 1999)

0

5

10

15

20

25

30

35

40

45

50

0 30 60 90 120

Mito. mem. potential (%)

Duration (minutes)

CONTROL

IGF

Effect of IGF-I on mitochondrial membrane potential

** *

Insulin-Like growth factor-I in buffalo seminal plasma

Average : 116.83 ± 28.34 ng/ml Range : 41.41 - 198.95 ng/ml

Effect of IGF-I on semen qualityPhysiologic al conc. of IGF-I :100 ng/ml

Frozen thawed Buffalo semen

Incubation-TRIS buffer at 37 deg C / 2 h (without egg yolk and glycerol)

Positive role of IGF-I on sperm motility, progressive forward motility, functional membrane integrity lipid peroxidation levels during 120 min study period in buffalo

Improvement of sperm functional parameters and fertility after addition of IGF-I may be due to reducing lipid peroxidation

IGF-I positively influences semen fertility