Advancements in quality assessment of frozen semen and correlations between lab tests and field fertility data

Advancements in quality assessment of frozen semen and correlations between lab tests and field fertility data

A. Kumaresan

Senior Scientist (Animal Reproduction),

Theriogenology Lab, National Dairy Research Institute, Karnal, Haryana


With the inception of cryopreservation of semen, transmission of superior genetic material by artificial insemination has become rapid and wide spread crossing the geographical boundaries. Advances in frozen semen technology have improved post thaw semen quality which is reflected in improved fertility with frozen thawed semen over time. However, precise determination of the quality of the genetic material before dissemination is of utmost importance to fully exploit the technology of cryopreservation. In spite of several developments in the semen freezing and thawing, approximately 50% of sperm are rendered immotile by cryopreservation and fertilizing capacity of spermatozoa is significantly decreased (Watson et al., 2000) leading to decreased conception rates with frozen semen compared to fresh semen. Therefore determining the quality of the frozen semen through certain in vitro test in relation to fertility is of prime importance in the dairy animal breeding industry.

So far post thaw motility of semen has been considered as an indicator of fertilizing ability  of  the  spermatozoa  and  it  is  mostly  used  for  quality  control  in  breeding industry. Though good post thaw motility is prerequisite for fertilizing ability of spermatozoa, it is not the sole attribute responsible for fertility. Attaining conception depends on structural and functional integrity of the spermatozoa. In addition to motility estimation several other tests such as viability estimation, morphology, acrosome integrity, plasma membrane integrity etc. also been incorporated for quality control of semen over the years. With the advent of fluorescent microscopy, flowcytometry and advances in molecular biology, now it has become possible to evaluate the spermatozoa in terms of specific functions that are well related to fertility. However, till date there is no reliable test for detecting the fertilizing potential. This lack of consistency can be ascribed to the multi factorial mature of the fertilizing ability  of  spermatozoa  and  lack  of  accurate  data  correlating  fertility  with  sperm function tests. There is ardent need for developing in vitro tests or battery of tests which can rapidly and inexpensively determine the quality of the semen in relation of fertility. Keeping view of these intricacies present article discusses different available quality control tests in relation to fertility and recent developments in quality control test of bovine semen.

Quality control of frozen semen - why it is critical?

To achieve high fertility, sperm must be motile, retain their ability to produce energy via metabolism in mitochondria, maintain normal plasma membrane configuration and   integrity,   intact   acrosome   membrane,   intact   receptors   that   permit   the spermatozoa to bind to the zona pellucida, maintain enzymes within the acrosome to allow penetration of ova and a nucleus capable of decondesation (Graham and Moce, 2005). Disruption of any of these functions or abilities will significantly affect the sperm’s ability to achieve fertilization. Cryopreservation of spermatozoa is a damaging phenomenon. Various sperm membranes and organelles are compromised by cryopreservation. Induction of premature capacitation and acrosomal reaction, altered mitochondrial function, reduced motility and failure of chromatin decondensation, influence the viability and fertility of the sperm cells (Bailey et al, 2003).  Cholesterol efflux from sperm and influx of sodium and calcium ions impart capacitation-like changes  (“cryocapacitation”)  to  sperm.  Increasing  the  number  of  dead  cells  also releases reactive oxygen species (ROS) which is detrimental to the live sperm population.  Damage to the cellular membranes is of greatest consequence because it has a carry-over effect on other sperm structures and functions. All these damages together reflect in terms of reduced fertility with frozen thawed semen than fresh semen. Therefore determining the quality of the semen prior to use is of utmost interest for the success of artificial breeding programme.

Quality control of semen can be defined as single test or set of tests to determine structural and functional integrity of frozen thawed spermatozoa in order to determine the suitability of the dose to be used in artificial insemination.  Traditionally semen quality control involves subjective assessment of motility, sperm morphology assessment and an estimate of the concentration of spermatozoa (Gillan et al., 2008).

These tests determine minimum standard for the frozen semen to be used. However, they have limited value for predicting the fertility (Rodriguez-Martinez, 2000). As a result, attention has been directed towards the assessment of other aspects of semen quality as predictors of fertility, such as viability, acrosomal integrity, membrane status, capacitation status, lipid scrambling, mitochondrial potential, Ca2+ uptake, apoptosis, DNA integrity, membrane proteins and the ability of spermatozoa to swim- up. Few in vitro sperm parameters show a reliable and repeatable correlation with field fertility (Rodriguez-Martinez, 2000). With development in sperm cryobiology, in vitro fertilisation or combination of number of in vitro tests has been combined to produce predicting model. The predictability of the model will increase with the increased number of tests incorporated in the model.

Traditional  quality  control  tests  for  frozen  semen  and  their  relation  with fertility:

Since the inception of frozen semen technology, the search is on for a proper marker for fertility of the frozen semen. Several tests has been developed and correlated with fertility. Few of them become regular quality control test for measuring suitability of the frozen semen doses for artificial insemination.

1. Post thaw motility: The most widely used quality control of frozen semen is done through visual observation of percentage of motile spermatozoa in a semen sample. This has become popular laboratory as it is quick, easy to perform and correlated with fertility. The most critical part of the test is the subjectivity and depends on the expertise of the evaluator. The error can be reduced to some extent by multiple estimations to minimize sampling error. The subjective biasness of the assay is somehow reduced by introduction of computer assisted semen analyzer (CASA), time-lapse photography using conventional or digital cameras, laser-Doppler spectroscopy or photometric methods. Among them CASA has emerged as a reliable measure of motility parameters of spermatozoa with accuracy and repeatability. This has also added the advantage of estimating many other kinematic parameters and sub population analysis. Although a very important assay, motility estimation evaluates only one spermatozoal attribute necessary to reach the site of fertilization and to fertilize an oocyte, and therefore, are not consistently highly correlated with fertility. The correlation between percentages of motile or progressively motile sperm in a semen sample and fertility has varied between 0.015–0.84 among different studies (Graham et al., 1980).

2. Sperm  concentration:  Number  of  spermatozoa  in  semen  dose  is  critical  in attaining fertility with frozen semen. The required number of spermatozoa in semen doses varies with standards of the country and ranged between 10-20 million spermatozoa/ frozen semen doses. The concept behind maintaining specific concentration is that there is need of certain number for synergistic facilitation of spermatozoa  movement  in  female  reproductive  tract.  Traditionally,  the concentration is measured using Haemocytometer or Makler counting chamber after diluting the semen. Sample dilution, charging of the sample in counting chamber and finally counting are the possible point of possible error introduction. With the introduction of spectrophotometry based concentration measurement with automatic dilution apparatus, measuring concentration has become more accurate.

3. Sperm morphology: Semen doses possessing greater number of morphologically abnormal spermatozoa have lesser chances of fertilizing the oocyte. Studies indicate that semen doses with higher percentage of morphologically abnormal sperm lead to reduced fertility (Lavara et al., 2005). Morphology evaluations can be conducted visually or using automated image based morphometry. The morphological abnormality has also been further classified into major/ primary (acquired in the testis during spermatogenesis) and minor/ secondary (acquired after spermatozoa left testis) abnormality considering the part of the spermatozoa which is abnormal and type of abnormality. Although sperm morphology affects fertility, some of these traits appear to be compensable, meaning that fertility can be improved if more sperm are inseminated, while others are uncompensable (Walters et al., 2005), making correlations between morphology evaluations and fertility difficult.

4. Incubation/   thermo   resistance   test:   Sustaining   the   motility   in   female reproductive organ is prerequisite to attain successful fertilization. This prerequisite is mimicked in the incubation or thermo resistance test and spermatozoa are subjected  to  incubation  in  370C.  The  viability  reflected  in  terms  of  motile spermatozoa is a good indicator of in vivo viability. The ease of performing the assay has made this a choice of test for quality control of frozen semen.

5. Acrosomal integrity: A spermatozoa must maintain an intact acrosome up to the time it binds to the zona pellucida of the oocyte and undergoes the acrosome reaction, which releases the acrosomal enzymes permitting the sperm to digest a hole through the zona pellucida, thereby allowing the spermatozoa access to the oolemma (Yanagimachi et al., 1981). Structural and functional intactness of acrosome is one of the prerequisite to attain high fertility. There is damage of acrosome during cryopreservation due to physical wear and tear. Therefore measuring the acrosome intactness has become a useful tool to estimate the fertilizing ability. The acrosome integrity is traditionally measured using Giemsa staining  (Yanagimachi,  1994).  Studies  have  reported  high  correlation  between fertility and percentage of spermatozoa with normal acrosome (0.97, Kumaresan et al, 2001). The limitations with the assay labour intensiveness and fixing a& staining requires expertise to avoid artifact.

6. Plasma membrane integrity: The integrity of the sperm plasma membrane is often synonymous with the viability of the spermatozoa though it is  actually assess whether or not the cell plasma membrane is intact or not. In order to fertilize an oocyte, a sperm must have an intact and competent plasma membrane. Destabilization of the plasma membrane occurs during lowering of temperature during cryopreservation. The spermatozoa undergo important events in the female reproductive  tract  like  capacitation  and  acrosome  reaction  to  attain  fertilizing ability.  For  these  events  to  take  place  the  spermatozoa  must  maintain  the functional integrity of cell membrane. Classically plasma membrane integrity is measured using eosin-nigrosin or eosin aniline blue which bind to the DNA of the sperm if the membrane is damaged. Unlike an intact sperm plasma membrane (‘live’), which is not permeable to eosin, a sperm with damaged cell membranes (‘dead’) takes up the red dye and becomes colored . In the one step eosin-nigrosin stain, nigrosin is used to provide the necessary background contrast for the live sperm cells, which remain unstained (Ramalho-Santos et al., 2007).

Hypo-osmotic  swelling  test  is  also  used  to  determine  the  plasma  membrane integrity. For this assay, sperm are incubated in a hypoosmotic medium, and then assayed, using light microscopy. Due to hypo osmotic medium outside there will be influx of fluid inside spermatozoa. If the plasma membrane over the principle piece is intact, the membrane will swell causing the tail to coil, while spermatozoa with damaged principle piece membranes will not swell. Though the test is highly correlated with the viability of spermatozoa, in cattle, a preliminary study observed that the osmotic swelling resistance test was not related to IVF success (Rota et al., 2000).

7. Bovine cervical mucus penetration test: Spermatozoa have to traverse through the different mucus of female reproductive tract to reach to the site of fertilization. The ability of the spermatozoa to pass through the mucus membrane is a prerequisite for fertilization. Therefore measuring the capability of the spermatozoa to penetrate through the cervical mucus of estrus cows is representative of its ability to traverse through female reproductive tract in vivo. The spermatozoa are incubated for an hour in 370C with cervical mucus in capillary tubes and observed under phase contrast microscope to measure the distance covered by the vanguard spermatozoa. The test is sometime constrained by the quality of the cervical mucus which is further overcome by using artificial cervical mucus.

8. Microbial load estimation: Disseminating disease free frozen semen doses for artificial insemination is prerequisite for its success and therefore estimation of microbial load in the frozen semen dose is also considered as quality control test for cryopreserved semen. Standard plate count using nutrient agar is regularly used for microbial load assessment. The detailed procedure and relationship with fertility is out of the scope of the paper and hence not discussed in detail.

Table 1: Correlation between traditional quality control assay and fertility


Name of the assay





Post thaw motility







Foote et al., 2003; Gillan et al., 2008

Incubation/ thermo resistance test




Moce and Graham, 2008


Acrosomal integrity




Saacke and White, 1972


Kumaresan et al (2001)


Plasma membrane integrity


0.12 - 0.635

Moce and Graham, 2008; Gillan et al., 2008






Moce and Graham, 2008


Kumaresan et al (2001)

Bovine cervical mucus penetration test




Gillan et al., 2008

The need of replacement – scope for advancement

Spermatozoa should be structurally and functionally at optimum level for attaining successful conception. The traditional quality control tests for frozen semen mostly focussed only on structural integrity of the spermatozoa, which led to lower correlation with fertility. There are several other reasons which led to search of novel and reliable test for quality control of frozen semen.

1. Traditional tests mainly focused on structural integrity and more specifically on motility and membrane related parameters.

2. Provide no insight on the functional integrity of spermatozoa which are rather more importance to attaining conception.

3. Mostly subjective and carry biasness between evaluators

4. Staining procedures are cumbersome and need expertise personnel to evaluate.

5. Labour intensive and time consuming.

This made the avenue for further development of quality control tests for spermatozoa which may able to predict the functionality of the spermatozoa so as to predict fertilizing ability more appropriately. Several fertility assays have been designed to measure the ability of spermatozoa to accomplish specific steps in the fertilization process, rather than measuring more general characteristics of spermatozoa. These assays are referred to as function based assays. The advances in terms of fluorescence microscopy, flow cytometry and molecular biology have provided more advanced and accurate tools to rapid detection of functional and structural assessment of spermatozoa.  Development in flow cytometry has made the quality control measures more efficient and quick with accuracy

Advanced QC tests for frozen semen and their correlation with fertility

Several assays has been developed in last few decades to determine the quality of the frozen semen sample and checked against the fertility results. Among them few important assays which are important considering the function or structure it is measuring is discussed in this part.

1. Plasma  membrane  integrity:  For  this  process,  combination  of  DNA-binding fluorescent stains is used (SYBR 14 or CFDA along with Propidium iodide). SYBR 14 or CFDA dead is membrane permeable stain and stain all the sperms green irrespective of live or dead and Propidium can penetrate only damaged membrane and stain them red (Garner, 1994). Sperm showing yellow or orange staining are designated as moribund sperm (Ramalho-Santos et al., 2007). The fluorescence can be measured in fluorescence microscope by manual counting of cells which can be further mechanized by using flow cytometer where more number of cells can be counted in short period of time.

2. Acrosome integrity: The acrosome integrity is measured using FITC-PSA or FITC- PNA (Pisum sativum agglutinin linked to fluorescein isothiocyanate or Lectin from Arachis hypogaea (peanut) conjugated fluorescein isothiocyanate), which binds to the acrosomal content of sperm after plasma membrane permeabilization, thus determining  the  presence  or  absence  of  the  acrosomal  matrix  (Gamboa  and Ramalho-Santos, 2005, Gadella, et al., 2008).

3. Capacitation status (CTC):    Studying  capacitation  status  is  also  of  immense interest as it is major determinant of fertilizing ability of sperm. Sperm capacitation involves physiological and functional alterations, such as intracellular calcium increase, cholesterol efflux from the plasma membrane, sperm pH and protein phosphorylation (Rathi et al., 2001, Naz and Rajesh, 2004). The capacitation status of spermatozoa can be detected by using Chlortetracycline (CTC) staining. CTC binds with Ca2+  and therefore indicative of increased intracellular calcium thereby capacitation. Three patterns can be observed with CTC staining (Shi et al., 1997) viz. pattern F, which consists of fluorescence over the entire head and equatorial region, is characteristic of uncapacitated acrosome-intact sperm; pattern B, which consists of a fluorescence-free band in the post-acrosomal region, represents capacitated acrosome-intact sperm; and pattern AR (Acrosome reacted), which consists of very low fluorescence over the head, corresponds to sperm that have undergone acrosomal exocytosis. Capacitation status of spermatozoa is highly correlated with fertility, less number of spermatozoa capacitated in a semen dose favours higher fertility.

Protein phosphorylation can be monitored by the detection of phosphotyrosine (residues  in  sperm,  which  can  be  assessed  by  immunocytochemistry.  This technique  allows  the  detection  and  localization  of  protein(s)  using  specific antibodies (Ramalho-Santos et al., 2004). First a primary antibody specifically recognizes and binds to the protein to be detected. In order to visualize the protein using epifluorescence microscopy a secondary antibody tagged with a fluorescent tag, and which is specific for the primary antibody, is then used. Alternatively the primary antibody may be itself fluorescently tagged. The tyrosine phosphorylation can also be detected using western blotting technique. In Western blotting the secondary antibody can be detected using chemilimiscent reaction, infra red tag or fluorescent tag

4. Membrane scrambling: Sperm membrane like other membranes comprise of lipid protein lipid bilayer. Apart from major bilayer forming lipid, there are alternative minor aggregate of non bilayer forming lipids in hexagonal form. This lipids form a tight seal around the protein lipid transition areas in membrane by forming annular ring around the integral proteins and thereby stabilizing the bilayer configuration (Jain, 1988). At the time of temperature drop during liquid storage in cold temperature or cryopreservation, lipids undergo thermotropic phase transition from liquid crystalline to gel phase and begin to aggregate. As the phase transition temperature is higher in non bilayer forming lipids, they undergo phase transition before the bilayer forming lipids leaving the bilayer forming lipids. On thawing or rewarming, these non bilayer forming lipids do not revert back to their original protein association causing membrane scrambling and loss of selectively permeable property (Quinn, 1989). Apart from this there is loss of membrane phospholipids during cold shock which skew the ratio between cholesterol and phospholipid. Membrane scrambling precedes capacitation in spermatozoa and thus measuring the percentage of spermatozoa with membrane lipid scrambled is indicative of spermatozoa quality.

Membrane scrambling is detected by using Merocyanine 540 staining. It is a useful fluorescent hydrophobic probe for lipid packing because it binds preferentially to membranes  with  highly  scrambled  lipids.  It  is  also  sensitive  to  heat-induced changes in the organization of membrane lipids, thus allowing the monitoring of heat inflicted alterations in the lipid architecture of the cells (Rathi et al., 2001).

5. Mitochondrial membrane potential: Mitochondria are responsible for supplying energy to the spermatozoa for its motility. Mitochondrial membrane potential (transmembrane electrical gradient) is also subjected to alteration with altered proteomics structure during cryopreservation and of utmost interest of study as a biomarker of mitochondrial functionality. It has been reported that mitochondrial membrane potential is highly correlated with motility and fertilizing ability of the mammalian sperm (Ramalho-Santos et al., 2007; Paoli et al., 2011).

The lipophilic cationic dye, JC-1 (5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’ tetra ethyl benzimidazolyl carbocyanine iodide), accumulates in mitochondria depending upon membrane potential. An increase in mitochondrial membrane potential, and thus an   increase   in   intra-mitochondrial   JC-1   accumulation,   is   indicated   by   a fluorescence emission shift (from green to red). This happens due to the formation of probe aggregates in highly active mitochondria, with a concomitant shift in fluorescence emission. On the other hand, low membrane potential mitochondria accumulate  JC-1  in  its  monomeric  form,  and  thus  exhibit  green  fluorescence (Ramalho-Santos et al., 2007).

Sperm mitochondrial function can also be assessed using fluorescent vital dyes, such as Mito Tracker Green. Mito Tracker Green is readily sequestered in mitochondria with a high mitochondrial potential, and the mid piece of spermatozoa with active mitochondria presents green fluorescence, while the mid piece of spermatozoa with non-functional mitochondria emits no fluorescence

6. Spermatozoal apoptosis detection: The presence of putative apoptotic markers on cryopreserved spermatozoa is of interest in terms of addressing the quality of a semen sample. It can be monitored using the fluorescence assay involving the phosphatidytlserine binding protein Annexin V. Annexin V is a Ca2+-dependent phospholipid binding-protein that has a high affinity for PS, and can serve as an early apoptosis marker. In this case Annexin V is tagged with the fluorescent moiety Alexa Fluor 568 (red fluorescence). Annexin V is usually used together with nuclear stains such as Syto 17, Hoechst 33342 etc., which serve as markers for cell permeability and thereby viability

7. Spermatozoal DNA integrity: Integrity of the genetic material is of concern for finally fertilizing the oocytes. Several assays in this regard has been developed to determine the integrity of chromatin of spermatozoa.

Chromomycin A3 (CMA3) is a guanine cytosine specific fluorochrome that reveals chromatin that is poorly packaged in human spermatozoa via indirect visualization of protamine deficient DNA. Chromomycin A3 and protamines compete for the same binding sites on spermatozoa. Spermatozoal chromatin structure is considered an uncompensable trait because defects in spermatogenesis and spermatozoal maturation that cause aberrant spermatozoal chromatin structure may not be manifested until spermatozoa oocyte fusion, egg activation, or early development (Evenson, 1999). Evaluation of spermatozoal chromatin structure has also been highly correlated to fertility in cattle (Ballachey et al., 1988). Comet assay is also used to determine the gentic integrity of the frozen semen sample  and  emerged  as  quality  control  assay  of  the  frozen  semen.  It  is  a microscopic assay, in which sperm are embedded in agarose on a glass slide, lysed, exposed to electrophoresis, stained with ethidium bromide and observed under fluorescence microscope to determine the degree of migration of single and double DNA strand break fragments. Sperm possessing more DNA damage exhibit larger DNA migration areas than sperm possessing little DNA damage (Evenson andWixon, 2006)

The Sperm chromatin structure analysis (SCSA) assay, evaluates the amount of damage  sustained  to  sperm  DNA  after  low  pH  treatment  to  induce  DNA denaturation. The sperm are then stained with acridine orange, which stains native DNA green and denatured DNA orange. The ratio of the amount of red to green for each individual spermatozoa is assessed by flow cytometry, with sperm containing greater red to green ratios exhibiting more DNA denaturation than sperm exhibiting lesser red to green ratios (Evenson and Wixon, 2006). The assay becomes popular considering  the  ease  of  performing,  quickness  and  volume  of  sperm  can  be evaluated. The terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay is one of the more known assays for DNA damage. In essence this method transfers a labeled nucleotide (BrdUTP) to the 3 OH group of a damaged DNA strand, a reaction catalyzed by a terminal deoxynucleotidyl transferase. The modified nucleotide is then detected using an appropriate antibody directly labeled with a fluorescent tag. Sperm with damaged DNA are thus fluorescent.

8. Detection of Calcium level (Fluo 4): Several intracellular changes are known to occur  during  capacitation,  including  increased  membrane  fluidity,  cholesterol efflux, intracellular Ca2+ concentrations, changes in motility patterns, and protein tyrosine phosphorylation (Breitbart and Naor 1999). Given that an increase in the concentration of calcium in sperm cells has been demonstrated during capacitation in several mammalian species it could be speculated that such changes may also occur during cryocapacitation. In support of this theory, it has been shown that the levels of (Ca2+) were higher in cryopreserved than in fresh bovine spermatozoa (Collin and Bailey 1999). Sperm calcium level is measure using Flou 4, Fluo 3 or Indo 1 fluorescent stain which is binds specifically with calcium. The stain is used along with Hoechst 33342 in order to differentiate between membrane permeable or non permeable spermatozoa. This combination also helps to avoid counting of debris particles during using flowcytometry.

9. Detection of Reactive oxygen species production: Reactive oxygen species (ROS), specifically superoxide anion (O2-), hydrogen peroxide (H2O2), and nitric oxide (NO), play a role in the cascade of events leading to capacitation (Aitken, 1995, Belen et al., 2000). In cells, ROS can be produced by intracellular oxidases and peroxidases or by other enzymes such as Cytochrome p450 or nitric oxidase synthase, though the main source is considered as leakage of electrons from the electron transport chains (Ford, 2004).        The release of this oxidase from dead sperm in egg yolk extender reduces the motility and viability of the remaining living bull sperm. Contamination of leukocytes also acts as prolific source of ROS production (Babior et al., 1997). When the balance between ROS production and detoxification by antioxidants is disrupted, an excess of ROS creates oxidative stress. ROS such as H2O2  are known to arrest motility and block oxidative metabolism in sperm. Also, ROS decrease oocyte penetration by sperm and block sperm-egg fusion (Mammoto et al., 1996). Sperm DNA damage by ROS also has been reported, which has serious consequences on post fertilization development (Aitken et al., 1998). Lipid peroxidation in bull sperm increases after cryopreservation. Moreover, frozen-thawed bull sperm are more easily peroxidized than fresh sperm. Unsaturated fatty acids within the sperm cell membrane are mostly susceptible for this type of peroxidation damage which destabilizes the membrane. Therefore detection of ROS level in frozen semen sample can be correlated with fertility. The reports correlating ROS level with fertility level is scanty and need more concrete data to establish the relation.

Single test versus a battery of test

Fertility is multi-factorial and attaining a successful conception depends on proper functioning of all of the steps leading to fertilization. Therefore merely measuring a single attribute cannot be predictive of the fertility of frozen hawed sample. Moreover correlations between laboratory results and fertility are inconsistent between studies. This made the decision of choosing appropriate quality control test for frozen semen more critical. Few points which can be mentioned in this regard which is responsible for making the decision tough –

i. Sperm must possess many of the functional and structural attributes to fertilize oocytes and not merely one or two attributes.

ii. For most attributes, there is no minimum or maximum level rather a spermatozoa must possess a sufficient amount of each attribute to fertilize an oocytes. Thus it is easy to propose that which sample is going to have poor fertilizing capacity but it is tough to judge which sample is of high fertility.

iii.In addition there are many variables in part of female and handling of frozen semen sample which lead to poor fertility.

iv. Finally  accuracy  of  fertility  estimation  is  dependent  upon  having  sufficient numbers of inseminations from a particular semen sample


Fertilization is still full of mystery and there is many part is still unknown than what we know so far. Advancement of scientific knowhow has unravelled many aspects of fertilization and helped to develop new quality control tests.  Now-a-days fluorescent staining based tests to evaluate sperm functions that are directly related to fertility are available and can be performed easily at semen stations. Thus instead of continuing the traditional routine semen analysis tests, incorporation of advanced tests, using specific probes to assess specific functions related to fertilizing potential of spermatozoa, into the routines of semen stations is expected to yield good results in terms of identifying fertility potential of the bulls. Once incorporated into the semen stations, it is also possible to grade and sell the superior quality semen in a premium price, which would not only benefit the semen stations but also the end users in terms of improved fertility.


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Dr. A. Kumaresan


Completed  his  Bachelor’s  degree  in  Veterinary  Science  from  Madras  Veterinary College, TANUVAS. He received Master’s degree and Ph.D from Indian Veterinary Research Institute, Bareilly. He received a diploma in Reproductive Endocrinology from Uppsala University, Sweden and he is a Post Doc from Swedish Agricultural University.

Current Professional Engagement:

Senior  Scientist  (Animal  Reproduction),  Theriogenology  Lab,  Livestock  Research Centre, National Dairy Research Institute, Karnal.

Professional Experiences:

Started his professional career in Animal Reproduction in National Dairy Development Board. In Indian Council for Agricultural Research, he had worked in various research institutions as Scientist conducting pioneering research. Worked extensively on Male fertility markers, Semen Biology, Cryopreservation of semen; Fertility improvement in male and female farm animals and Livestock production systems. Standardized the method of extraction of testicular cells (Precutaneous needle aspiration biopsy - PNAB) from  live  bulls  and  technique  of  bull  fertility  evaluation  based  on  testicular cell indices.    As  a  renowned  Scientist  in  reproductive  technologies  in  India  he  has authored more than 130  articles and 13 books. He has been a recipient of various fellowships and Lal Bahadur Shastri Young Scientist Award of ICAR for the Biennium, 2005-06.