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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
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
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
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
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
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
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).
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.
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.
In vitro fertility tests
Cervical mucus penetration testSperm-oocyte binding assayIn vitro sperm penetration assayCleavage rate
Disadvantage2. Handling- Many times-Loss of oocytes3. Staining is must4. Block to polyspermy-Mature oocyte
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
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
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