Impact of 24-h Cooling Prior to Freezing on the Survival of Domestic Cat (Felis catus) Epididymal Sperm

The aim of this study was to investigate the impact of a 24-h cooling period prior to freezing on domestic cat epididymal sperm viability. Fifteen tomcats were submitted to routine orchiectomy and sperm samples were retrieved from both epididymides in a Tris–glucose–20% egg yolk extender. For each tomcat, the diluted sperm was split into two equal volumes and cooled to 5°C at a rate of 0.5°C/min; one sample for 60 min (control) and the other for 24 h (cooled). After the cooling period, samples from both groups were frozen using an identical freezing protocol. Sperm samples were evaluated in three different periods: immediately after harvesting, after cooling at 5°C for 24 h (cooled group) and after freezing–thawing of control and cooled groups. Evaluations consisted of sperm motility and progressive status, sperm morphology and plasma membrane integrity (PMI) using two fluorescent probes. After cooling for 24 h, a decrease (p < 0.05) in sperm motility, progressive status and PMI was observed when compared to sperm samples immediately after collection. Comparing the results obtained after thawing, no difference (p < 0.05) was found regarding sperm motility, progressive status, PMI and sperm morphology between control and cooled groups. The results from the present study show that cooling cat epididymal spermatozoa at 5°C for 24 h prior to freezing does not lead to major damage of spermatozoa impairing the freeze–thaw process.     

Retinal Epithelial Fine Structure in the Domestic Cat (Felis catus)

The morphology of the retinal epithelium (RPE) and closely associated choriocapillaris and Bruch's membrane (complexus basalis) has been investigated in the eye of the domestic cat (felis catus) by light and electron microscopy. The RPE consists of a single layer of cuboidal cells joined laterally by apically-located junctional complexes. Basally (sclerally) these cells display numerous infoldings while apically (vitreally) two distinct types of processes enclose photoreceptor outer segments. Internally the large vesicular nucleus is centrally located. Smooth endoplasmic reticulum, mitochondria, polysomes and lysosome-like bodies are abundant. Rough endoplasmic reticulum while present is not plentiful. Over the tapetum lucidum, melanosomes are absent from the RPE cells while in non-tapetal locations melanosomes are large and plentiful. The endothelium of the choriocapillaris is highly fenestrated and these capillaries are deeply indented into the epithelial layer over the tapetum. Bruch's membrane (complexus basalis) is non-tapetal regions is the typical pentalaminate structure noted for most mammals. Over the tapetum however it is reduced to a trilaminate structure and when associated with the indented capillary profiles is further reduced to a single thickened basal lamina.     

Fine Structure of the Retinal Photoreceptors of the Domestic Cat (Felis catus)

The morphology of the retinal photoreceptors of the domestic cat (Felis catus) has been investigated by light and electron microscopy. In this species the photoreceptors can be readily differentiated and adequately described by the classical terminology of rods and cones, with rods being the more numerous. Rods are long slender cells with inner and outer segments of much the same diameter. Cones are shorter cells with a conical outer segment that tapers distally and an inner segment which is wider than the outer segment. Both rods and cones are highly polarized and compartmentalized cells and consist of an outer segment, a connecting cilium, an inner segment, a nuclear region and a synaptic process leading to an expanded synaptic ending. Morphological similarities and differences are noted between the rods and cones for the various regions of these cells.     

Assessment of Selected Quality Parameters of Epididymal Cat (Felis catus s. domestica,L. 1758) Sperm Using Flow Cytometry Method and Computer Assisted Sperm Analyser

The improvement of biotechnical methods connected with fast and precise semen quality assessment and its utilization in assisted reproductive techniques is an urgent necessity in felids. The aim of this study was to evaluate some quality parameters (i.e. the viability and share of cells with intact plasma membrane) of epididymal sperm of cats using the flow cytometry method and computer‐assisted sperm analysis (CASA) examination. The material consisted of epididymal spermatozoa flushed from 22 pairs of epididymes after routine neutering procedures obtained from domestic cats aged between 8 and 36 months. The epididymes were cut and incubated with an extender without egg yolk. The samples were assessed for sperm viability (Live/Dead Sperm Viability Kit^®), percentage of subtle membrane changes (Apoptosis Detection Kit^®) and motility using FACScalibur flow cytometer and assisted sperm analyser htm ivos version 12.2. The flow cytometry method revealed 71.3% and 84.4% of live sperm using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit respectively. The population of early‐apoptotic and late‐apoptotic sperm were 0.8% and 1.1% respectively. The CASA examination found 51.5% of motile sperm. However, the motility examination under light microscope revealed 69.5% of motile sperm. The data revealed an indistinctive per cent of apoptotic cells and 18.9% and 15.6% of dead cells using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit, respectively, which indicate that the sperm obtained after flushing the epididymis possess potential properties for further assisted reproduction techniques.     

Vascularization and Production of Mitogenic Factor(S) in Domestic Cat (Felis catus) Testes: Preliminary Results

Some male seasonal breeders are known to undergo testicular growth and regression throughout the year. The periodic and fast physiological development and regression processes, with simultaneous fast changes in blood flow, appears to be stimulated by angiogenic substances and inhibited by anti-angiogenic factors. The objective of this study was to evaluate the effect of season on microvascularization and production of angiogenic factors by testicular tissue, using the male cat as a model. Monthly, testes were collected post mortem from domestic stray cats, for histology and for tissue culture. Testis explants were cultured for 18h and conditioned media were tested for their ability to stimulate mitogenesis of bovine aortic endothelial cells (BAEC). Vascular endothelial growth factor (VEGF) was used as a positive control. Proliferative response of BAEC to samples was evaluated by determining the number of cells in each well using a Neubauer chamber. Vascular density was assessed by a computerized image analysis ('Scion Image, NIH, USA') based on the total histological area occupied by the vascular lumen. Percentage data subjected to arcsine transformation were analysed by one-way ANOVA and post-comparison tests. There was a significant increase in microvascular areas in November ( P  < 0.001), when compared to the remaining months (LSD test). All cat testicular tissue showed the capability to increase BAEC proliferation, when compared to negative controls, throughout the study. However, there was an increase in BAEC mitogenesis in February ( P  < 0.05) and November ( P  < 0.001), when compared to the other months. Our data show that cat testicular tissue from different periods of the year stimulates angiogenic factor(s). Furthermore, changes in angiogenesis may play a role on vascular growth and regression of the testes during the breeding and non-breeding season in the male cat.     

Serum Starvation and Full Confluency for Cell Cycle Synchronization of Domestic Cat (Felis catus) Foetal Fibroblasts

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p ≤ 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p ≤ 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.     

Expression of Survivin,Bcl‐2 and Bax Proteins in the Domestic Cat (Felis catus) Endometrium During the Oestrus Cycle

Apoptosis has been shown to be an important regulator of endometrium function. To clarify the regulation of apoptosis in the cat endometrium during the normal oestrus cycle, the expressions of the apoptosis‐related proteins (Bcl‐2 and Bax) and their correlation to the inhibitor of apoptosis protein Survivin were analysed using immunohistochemistry. The TUNEL technique (TdT‐mediated dUTP nick end labelling) was also used to detect DNA fragmentation characteristic of apoptotic cells. The results demonstrated that TUNEL labelling is not effective for the detection of apoptosis in cat endometrium. Survivin was expressed in the luminal and glandular epithelial cells of cat endometrium during all phases of the oestrus cycle. Survivin was localized in both the cytoplasm and nuclei of superficial and deep uterine gland cells during the luteal phase, while only cytoplasmic staining was observed during the follicular and anoestrus phases. Bax immunoreactivity in the cytoplasm of luminal and glandular epithelial cells as well as the smooth muscle cells of blood vessels was weak in the anoestrus phase. Compared with anoestrus, the intensity of Bax immunostaining was moderate in the follicular phase and increased dramatically in the luteal phase. Bcl‐2 immunostaining in the cytoplasm of luminal and glandular epithelial cells was moderate in the anoestrus phase. During the early follicular phase, cytoplasmic Bcl‐2 immunostaining was detected mostly in glandular epithelial cells. In the mid‐follicular phase, in glands, the amount of Bcl‐2 protein increased progressively from the superficial to the deep layer. In contrast, the expression of Bcl‐2 decreased in the secretory phase, being very low or absent in the mid‐ and late luteal phases. The overall results suggest that Survivin, Bax and Bcl‐2 proteins may cooperatively contribute to cell apoptosis and cell proliferation in the cat uterus during the oestrus cycle.     

Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus)

The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal donors is currently poor primarily, because of contamination with other testicular cells. This study aimed to purify SSC-like cells using different extracellular matrixes and a discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish. The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment 2). Experiment 3 was performed to test whether or not the additional step of Percoll gradient density centrifugation could really improve purification of SSC-like cells. Testicular histology revealed complete spermatogenesis with laminin expression essentially at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4 in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other incubation times and gelatin treatment (P<0.05). Percoll treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ± 0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51% for GFRα-1, P<0.05). These attached cells demonstrated a typical SSC-like cell morphology and also expressed POU5F1, RET and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient density centrifugation and laminin plating highly enriched the SSC-like cells population.     

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.     

The susceptibility of domestic cats (Felis catus) to experimental infection with Leishmania braziliensis

Over the last few years, several cases of feline leishmaniasis (FL) with cutaneous and visceral forms have been reported around the world. Nonetheless, the real susceptibility of cats to infection with Leishmania spp. and the outcome of leishmaniasis in these animals are poorly understood. Experimental studies on feline models will contribute to the knowledge of natural FL. Thus, in order to determine the susceptibility of domestic cats (Felis catus) to experimental infection with Leishmania braziliensis, 13 stray cats were infected with 10(7) promastigotes by the intradermal route in the ear and nose simultaneously and followed up for 72 weeks. Soon after infection, the earliest indication of a lesion was a papule on the ear at 2 weeks post-infection (w.p.i.). The emergence of satellite papules around the primary lesion was observed about 4 w.p.i. Two weeks later these papules coalesced and formed a huge and irregular nodule. Thereafter, there was lesion dissemination to the external and marginal surface of the ipsilateral ear, and later to the contralateral ear. At 10 w.p.i., some nodules became ulcerated. Nose lesions presented a similar evolution. At both sites, the largest lesion sizes occurred at 10 w.p.i. and started to decrease 15 days later. Ear and nose nodules healed at 32 and 40 w.p.i., respectively. Specific L. braziliensis IgG antibody titers (optical density> or = 0.01 as positive result) were detected as early as 2 w.p.i. (0.09 +/- 0.02) in only three animals (23%), and all cats had positive titers at 20 w.p.i. (0.34 +/- 0.06). Only three animals (38%) continued to show positive serology at 72 w.p.i. (0.08 +/- 0.02). Up to that time, none of the cats had lesion recurrence. In a feline model of cutaneous leishmaniasis, it seems that there is no correlation between active lesions and positive serology. The implications of these data are discussed.     

The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.     

The experimental transmission of various mycoplasmas of feline origin to domestic cats (Felis catus)

Mycoplasma felis and M. gatae recovered from domestic cats, M. arginini from a cheetah, an unidentified glucose-fermenting mycoplasma from a lion, and M. pulmonis from a rat were inoculated into specific pathogen free (SPF) kittens by the oral, ocular and nasal routes. During the 21-day experimental period a persistent infection was established in a proportion of the kittens challenged with M. felis, M. gatae and M. arginini. The mycoplasma isolated from the lion created a transitory infection in one kitten, but M. pulmonis could not be recovered even 24 hr after inoculation. None of the organisms investigated showed any pathological effect. The implications of this work in relation to the host-specificity and pathogenicity of the feline mycoplasmas are discussed. Résumé. On a inoculé à des chatons axéniques par les voies orales, oculaires et nasales, un Myco-plasmafelis et un M. gatae recueillis de chats domestiques, un M. arginini recueilli d'un guépard, un mycoplasme non identifié à glucose fermentant recueilli d'un lion et un M. pulmonis recueilli d'un rat. Durant la période expérimentale de 21 jours, on a établi une infection persistante dans une proportion des chatons soumis aux tests de Mycoplasma felis, de M. gatae et de M. agrinini. Le mycoplasma isolé recueilli du lion a créé une infection transitoire chez un chaton, mais le M. pulmonis n'a pu être recueilli, même 24 heures après l'inoculation. Aucun des organismes sous investigation n'a montré d'effets pathologiques. On a discuté des implications de ce travail en relation avec la spécifité d'hôtes et la pathogénicité de mycoplasmes félins. Zusammenfassung. Mycoplasma felis und M. gatae von Hauskatzen erlangt, M. arginini von einem Tschita, ein unidentifiziertes Glukose-fermentierendes Mycoplasma von einem Löwen, und M. pulmonis von einer Ratte wurden in keimfreie (SPF) junge Katzen geimpft, auf oralem, Augen- und Nasenweg. Während der 21 tägigen Versuchsperiode wurde eine anhaltende Infektion in den Katzen festgestellt, die dem M. felis, M. gatae und dem M. agrinini ausgesetzt waren. Das Mycoplasma von dem Löwen abgesondert, verursachte eine vorübergehende Infektion eines Kätzchens, aber M. pulmonis konnte selbst 24 Stunden nach der Impfung nicht gefunden werden. Keines der untersuchten Organismen zeigte irgendwelche pathologischen Effekte. Die Folgerungen dieser Arbeit im Verhältnis zu der Gast-Spezifität und Pathogenizität des Katzen-mycoplasmas sind diskutiert.     

Effects of Bovine Serum Albumin on Boar Sperm Quality During Liquid Storage at 17°C

This study aimed to investigate the effects of bovine serum albumin (BSA) on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 2, 3, 4, 5 and 6 g/l) of BSA, and sperm motility, plasma membrane integrity, acrosome integrity, total antioxidative capacity (T‐AOC) activity and malondialdehyde (MDA) content were measured and analysed. The results showed that Modena supplemented with 3, 4 and 5 g/l BSA could improve boar sperm motility, effective survival time and plasma membrane integrity (p < 0.05), decrease MDA content (p < 0.05), while no statistical difference was observed for sperm acrosome integrity and T‐AOC activity among these three groups (p > 0.05). The semen sample diluted with Modena containing 4 g/l BSA could achieve optimum effect, and sperm survival time was 7.5 days. After 7 days preservation, sperm motility, plasma membrane integrity and acrosome integrity were 54%, 49% and 78%, respectively. T‐AOC activity and MDA content were 1.03 U/ml and 17.5 nmol/ml, respectively. In conclusion, Modena supplemented with BSA reduced the oxidative stress and improved the sperm quality of boar semen during liquid storage at 17°C, and 4 g/l BSA was the optimum concentration. Further studies are required to obtain more concrete results on the determination of antioxidant capacities of BSA in liquid preserved boar semen.     

Apoptotic Changes in the Epithelium Germinativum of the Cat (Felis catus s. domestica, L. 1758) at Different Ages and Breeding Seasons

Apoptosis (programmed cell death) could be considered as a physiological process that takes part in a healthy organism, which helps to maintain organism homeostasis. The visible deterioration of semen quality and the number of germ cells is accompanied by a seasonal decrease of the reproductive activity in some species. This post-effect cascade is caused by apoptosis, which is the primary mechanism responsible for the elimination of germ cells during spermatogenesis. The aim of our study was to assess apoptotic changes in the epithelium germinativum in cat testes at different ages. One hundred and two pairs of testes were obtained from domestic cats aged between 4 months and 10 years. The paraffin-embedded tissue sections were labelled using the Oncogene and Calbiochem Research Products DNA Fragmentation Detection Kit (Cat# QIA21; Darmstadt, Germany), which allows the recognition of apoptotic nuclei in tissue sections with Fragment End Labelling (FragEL^TM) of DNA. The activity of apoptotic processes in cat testes collected from the spring-summer period compared with the autumn-winter season revealed that, 59.42% and 51.51%, respectively, males testes were characterized by insignificant changes. The obtained data revealed a distinctive apoptotic changes in the young animal testes before spermatogenesis onset. An intensification of programmed death cells in the epithelium germinativum in the elder cats (between 3–6 and 6–10 years) was not observed. Apoptotic changes slightly intensified in cats aged between 12 and 36 months.     

Effect of Cooling Rate to 5°C,Straw Size and Farm on Fertilizing Ability of Cryopreserved Rabbit Sperm

High fertility and prolificacy in rabbits are currently only achieved using fresh sperm. This study was conducted to determine if the cooling rate to 5°C, the straw size and the farm where artificial inseminations are performed have an impact on the fertilizing ability of rabbit sperm cryopreserved with an extender containing dimethyl sulphoxide (DMSO; 1.75 m ) and sucrose (0.05 m ). Slow cooling to 5°C improved neither fertility rate (58 vs 56% kindling rate for fast and slow cooling, respectively) nor prolificacy (6.5 vs 8.7 total born for slow and fast cooling, respectively; p < 0.05) compared to fast cooling rate to 5°C. The straw size did not have an effect on either fertility or prolificacy (47 vs 57% kindling rate and 6.3 vs 6.8 total born for sperm loaded into 0.25 and 0.5 ml straws, respectively). In addition, similar results were obtained between farms (46–57% kindling rate and 4.9–6.7 total born), although this effect should be studied further. In conclusion, with this extender, slow cooling does not present a beneficial effect on sperm fertilizing ability and either 0.25 or 0.5 ml straws can be used to freeze the sperm, obtaining similar results after artificial insemination. In addition, similar results were obtained between farms when using cryopreserved sperm, and these results were lower than those obtained after artificial insemination with fresh semen. Therefore, new approaches are needed to improve the results obtained when cryopreserved sperms are used before this type of sperm can be used for commercial purposes.     

Effect of astaxanthin on the quality of boar sperm stored at 17°C,incubated at 37°C or under in vitro conditions

The aim of the study was to investigate the effect of the antioxidant astaxanthin on boar semen. Twenty ejaculates from 10 boars (two ejaculates/boar) were extended and split in three groups: semen control (SC), solvent control (C; semen with dimethyl sulfoxide, the diluent of astaxanthin) and semen with astaxanthin (A) in concentration 0.5 μmol/L. Sperm quality parameters (motility and kinetics, morphology, viability, functional integrity of sperm plasma membrane by Hypo‐Osmotic Swelling Test [HOST] and DNA integrity) were assessed at 0, 24 and 48 hr of storage at 17°C (experiment I), before (0 hr) and after (1 hr) of sperm thermal resistance assay at 37°C (experiment II) and finally before (0 hr) and after (1 hr) sperm in vitro incubation (38.5°C, 5% CO₂, maximum humidity [experiment III]). In experiment I, group A performed overall better than group SC and as a tendency better than group C regarding viability. Total motility, rapid spermatozoa and HOST remained constant across time in group A, whereas they decreased in the remaining groups. In experiment II, regarding motility and viability, group A displayed better results across time than the other two groups. In experiment III, viability and total motility decreased in groups SC and C, while in group A, these parameters were not significantly different between the examination time points. In conclusion, astaxanthin has a beneficial and protective effect on boar semen quality under the investigated conditions.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.