Hyperactivated motility patterns of ram spermatozoa recovered from the oviducts of mated ewes

The contents of the oviducts of ewes were recovered by flushing with small volumes of culture medium, 22½–24¼ hr after mating. The ampulla was flushed separately from the uterotubal junction and isthmus. Among the motile spermatozoa recovered, a proportion exhibited “hyperactivated” motility, also known as “activated”, or “whiplash” motility. This was characterized by increased flexion of the neck, increased amplitude of the flagellar waves, and marked asymmetry of beat. Two types of hyperactivation appeared: in the first, spermatozoa swam in a repetitive, nonprogressive circling pattern and appeared to have intact acrosome caps; in the second, the spermatozoa showed a propensity to stick to glass by the equatorial segment and most had modified or missing acrosome caps. The proportions of motile spermatozoa exhibiting hyperactivation were greatest in the ampullae, as were the proportions with modified or absent acrosomes. Hyperactivation is a capacitation-associated phenomenon that has now been reported for one or more species from seven orders of eutherian mammals. It may well be a universal aspect of the prefertilization behavior of mammalian spermatozoa and is probably of advantage to the fertilizing spermatozoon within the oviduct.     

Calcium alters capacitation and progressive motility of uterine spermatozoa from +/+ and congenic tw32/+ mice.

The importance of calcium-dependent sperm processes for fertilization in vitro is well known, but their interaction with sperm transport in vivo is not yet clear. To determine whether exposure to calcium alters sperm physiology after incubation in the uterus, spermatozoa from +/+ mice were incubated in medium with 1.7 mM calcium prior to artificial insemination (AI). Spermatozoa from congenic tw32/+ mice were also tested because their flagella are hypersensitive to calcium. As a control, spermatozoa were incubated in calcium-deficient medium before AI. When recovered from the uterus 60 min post-AI, neither prior exposure to calcium nor genotype affected numbers of spermatozoa, or percentage of motile or acrosome-reacted spermatozoa. However, significantly more calcium-treated spermatozoa were capacitated and significantly fewer were progressively motile than spermatozoa preincubated without calcium. In addition, significantly fewer spermatozoa from tw32/+ mice than from +/+ mice were progressively motile. These results suggest that uterine sperm physiology is changed by prior exposure of sperm to calcium. Since the level of progressive motility of spermatozoa recovered from the uterus was correlated with their ability to reach the oviduct (as determined in a previous study), these data support the hypothesis that progressive motility of uterine spermatozoa is important for passage to the oviduct and fertility.     

Optimization of procedures for separation of motile and nonmotile bull and rabbit spermatozoa with bovine serum albumin gradients

The effect of equipment design, separatory media, and time and temperature of separation were studied. Discontinuous 4%/10% bovine serum albumin (BSA) gradients were used to isolate highly motile spermatozoa in rabbit and bull semen. For all conditions tested, motility of spermatozoa collected from the 4% BSA gradient layer (top) was less than or equal to the motility of the unseparated controls. Fractions collected from the 10% BSA gradient layer contained highly motile spermatozoa. In experiment 1, washed bull spermatozoa were diluted with phosphate-buffered saline (PBS) containing 2% BSA or 4% BSA before being fractionated on BSA columns contained in test tubes. Inclusion of BSA in PBS tended to reduce loss of motility during washing, but the proportion of sperm recovered was highest in PBS. In experiment 2, motility and recovery of buck spermatozoa collected from the 10% BSA gradient region tended to be higher when fractionation temperature was 30°C as compared to 35°C, and motility was significantly higher when incubation time was 30 min as compared to 1 hr. The proportion of sperm recovered was unaffected by incubation time. In experiments 1 and 2, 41 of 48 separations resulted in at least one fraction containing spermatozoa with motility greater than or equal to 90%. In the third experiment, the surface area on which bull and buck spermatozoa were layered was increased by forming the 4%/10% BSA gradients in conical supports. Separation of sperm on conically shaped columns was not as effective as on cylinders. The use of cylinders to support the BSA gradients and a separation time of 30 min at 30°C is recommended.     

Effect of ovulation and sperm motility on the migration of rabbit spermatozoa to the site of fertilization.

Few spermatozoa were present in the ampullae of females 12 h after intravaginal artificial insemination (AI) when there was no ovulation-inducing stimulus. When ovulation was induced, sperm distributions in the female tract 12 h after AI did not differ from those observed 12 h after natural mating. The number of spermatozoa in the oviductal isthmus was similar in all 3 groups as was the percentage of isthmic spermatozoa exhibiting 'activated' motility. When fertile mating was delayed for 8 or 12 h after coitus with a vasectomized male (i.e. 2 h before or after ovulation), spermatozoa were not present in the ampulla 4 h later. The numbers of spermatozoa recovered from the cranial isthmus after delayed matings and 12 h after natural matings did not differ, but after delayed matings the motility of isthmic spermatozoa was non-progressive or poorly progressive and none exhibited 'activated' motility. Flagellar activity of isthmic spermatozoa recovered 4 h after delayed matings and after natural matings was similarly depressed. These observations indicate that sperm ascent to the tubal ampulla in the sustained phase of transport, though enhanced by ovulation, must also depend on changes in flagellar activity and a specific pattern of motility, both of which appear only after spermatozoa have resided for more than 4 h in the female tract.     

Analysis of motility parameters from paddlefish and shovelnose sturgeon spermatozoa

Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s^-1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s^-1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.     

Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa

The possible role of tyrosine kinase in the regulation of fowl sperm motility was investigated by using a stable analogue of erbstatin, methyl 2,5-dihydroxycinnamate (2,5-MeC), a specific inhibitor of tyrosine kinase. This inhibited the motility of intact spermatozoa at 30°C in a dose-dependent manner. In contrast, the motility of demembranated spermatozoa was not inhibited by the same concentrations of 2,5-MeC. At 40°C, both intact and demembranated spermatozoa were almost immotile with or without 2,5-MeC. Additionally, intact spermatozoa, stimulated by the addition of Ca²⁺ or calyculin A, a specific inhibitor of protein phosphatases, lost their motility with the subsequent addition of 2,5-MeC at 40°C. However, unlike the motility, the ATP concentrations of spermatozoa were maintained in about 30–35 nmol ATP/10⁹ cells during these incubation periods. The activity of tyrosine kinase of spermatozoa at 30°C, estimated by measuring the phosphorylation of a synthetic peptide substrate, RR-SRC, was 0.17 pmol/min per milligram of protein. This activity was lower than that of fowl testes or chick brain but higher than that of chick liver. These results suggest that tyrosine kinase activity, which is not retained in the axoneme and/or accessory cytoskeletal components, may be involved in the maintenance of flagellar movement of fowl spermatozoa at 30°C. Mol. Reprod. Dev. 49:196–202, 1998. © 1998 Wiley-Liss, Inc.     

Effect of preservation of human semen sample at 4–6 and 25 °C on sperm motility

Sperm motility is the result of transverse movements that exist along its tail. It plays an important role in male fertility. The aim of this study was to evaluate the influence of keeping washed normozoospermic semen samples at 4–6 and 25 °C on the motility of spermatozoa. 26 semen samples of normozoospermic were washed twice in modified Ham’s F10 medium. Then, thirteen of the semen samples were kept in refrigerator (4–6 °C) and the remaining samples were stored in incubator (25 °C) for 12 days. On the 0 (immediately after sampling as control group), 1st, 2nd, 5th, 7th and the 12th days, the percentage of fast progressive (grade a), slow progressive (grade b), non-progressive (grade c) and immotile (grade d) sperm cells were calculated for each temperature. The data obtained from this study showed that the percentages of a, b and c grades of motile spermatozoa were significantly decreased (p?<?0.001) during 12 days at the both temperatures but reduction of these percentages has a gentle slope at 4–6 °C. There was no motile sperm after 12 days of storage. This study suggests that motile spermatozoa could be retrieved up to 7 days after the storage of washed normozoospermic men semen samples at 4–6 and 25 °C. Also, there were no motile sperm cells 12 days after sampling.     

The viability of hamster spermatozoa stored in the isthmus of the oviduct: the importance of sperm-epithelium contact for sperm survival

When hamsters mate shortly after the onset of estrus, spermatozoa are stored in the lower oviduct (isthmus) during the preovulatory period. The present study was performed to determine what proportion of the spermatozoa in the isthmus survive until fertilization. Females were mated 5 to 6.5 h before ovulation. When spermatozoa in the isthmus were observed through the wall of oviducts excised 2 h after the onset of mating, spermatozoa were seen free in the lumen, attached to the mucosal surface of the wall, and in crypts. The vast majority of spermatozoa in the lumen were immotile, whereas most of those attached to the mucosal surface of the wall and almost all of the those in the crypts exhibited flagellar movement. This suggested that attachment to the mucosa and/or storage in the crypts is beneficial to the survival of spermatozoa. Sequential flushing of an oviduct at various times (2-8 h) after mating was used to remove spermatozoa from the lumen (first flush), from the mucosal surface (second flush), and from the crypts (third flush). The highest number of spermatozoa was always contained in the first flush, the next highest in the second flush, and the smallest in the third flush. When Trypan blue was included in the flushing medium to differentiate live and dead spermatozoa, the first flush recovered the smallest percentage of liver spermatozoa (2-22%), the second flush slightly more (16-37%), and the third flush the highest (51-69%), regardless of the time after mating. These data indicate that the majority of spermatozoa stored in the hamster isthmus die before ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mink semen studies: I. Liquid preservation and prospect of freezing spermatozoa

Using 22 males, 41 semen samples were collected from the vagina of mink by means of a plastic tubing attached to a 1 ml syringe. Subsamples of vaginal semen were diluted in 4 different extenders, viz., tris (tris, citric acid, glycine, fructose, glycerol and egg yolk), PVP (tris extender with polyvinyl pyrrolidone and caproic acid), milk (boiled and filtered milk with glycerol) and sodium citrate. The extended semen samples were stored at 23, 5 and −196°C for varying periods and evaluated for % motile spermatozoa. In the tris extender storage for 3 days at 5°C or for 2 days at 23°C reduced the number of spermatozoa by more than 50%. When milk was used as the extender, the motility decreased from an initial value of 68% to 10% after 5 days of 5°C and to 8% after 4 days at 23°C. The PVP extender was not suitable for storage at any temperature. After being frozen at −196°C for 2 hr, the motility ranged from 3–10% in the tris extender and was zero in milk and PVP extenders. Prolonged storage for 7 days in tris extender reduced the motility to 1–7%.     

Stimulation of sperm motility and oxygen consumption of fowl spermatozoa by a low molecular weight fraction of seminal plasma

Washed fowl spermatozoa were incubated in a phosphate buffer containing various concentrations of fowl seminal plasma at 41 degrees C, normal body temperature, and the motility and oxygen consumption of spermatozoa were determined. Immediately after the incubation, spermatozoa showed good motility in the various diluents. However, with concentrations of seminal plasma at or below 20%, spermatozoa quickly became immotile. In contrast, at concentrations higher than 40% seminal plasma, spermatozoa were motile even after 15 min. As the concentration of seminal plasma was increased, oxygen consumption of spermatozoa also increased. A filtrate of the seminal plasma, obtained by passing the fluid through an Amicon YM-2 ultra-filtration membrane (Mr less than 1000), also stimulated the motility and oxygen consumption of spermatozoa. These results suggest that some low molecular weight factor(s) in fowl seminal plasma stimulated motility and oxygen consumption of fowl spermatozoa at 41 degrees C. A physiological role of this factor(s) may be to assist passage of spermatozoa through the vagina after natural mating.     

The movement characteristics of rabbit spermatozoa before and after activation

Spermatozoa were flushed with mineral oil from the lower isthmus of the rabbit oviduct at four hours postcoitus (pc) and 11 hours pc. Videotapes were made of sperm behavior in the native isthmic fluid and after dilution of the fluid with culture medium. The tapes showed that, initially, spermatozoa in the native isthmic fluid were virtually immotile, but immediately resumed movement on contact with the culture medium. Isthmic sperm motility then evolved over a five- to 10-minute interval into the characteristic biphasic pattern of activated movement. Cine films of isthmic spermatozoa taken with a high-speed camera were analyzed to determine flagellar beat frequency, maximum flagellar curvature, and swimming velocity. Progressiveness ratios and hydrodynamic power outputs were then calculated for individual spermatozoa. Two phases of activated sperm movement, a whiplash phase and a progressive phase, were identified and characterized. The power output of activated spermatozoa increased twentyfold in comparison with the preactivated state. The power output of activated spermatozoa did not differ between the two phases of activated movement.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Study of bovine sperm motility in shear-thinning viscoelastic fluids

To elucidate the process whereby sperm arrive at an egg in the female reproductive organs, it is essential to investigate how rheological properties of the fluid around mammalian spermatozoa affect their motility. We examined the motility and flagellar waveform of bovine sperm swimming in a fluid with similar rheological properties as mammalian cervical mucus. The results indicated that the surrounding rheological properties largely affected the flagellar waveform of mammalian spermatozoa; in particular, shear-thinning viscoelastic fluid increased the progressive motility of the sperm. To investigate the influence of flagellar waveform on sperm motility in more detail, the waveform was expressed as a function and the progressive thrust of the sperm was calculated based on the empirical resistive force theory. The results of this study showed that the progressive thrust increased with the curvature of the flagellar tip. Moreover, we calculated the thrust efficiency of motile sperm. Results showed that the thrust efficiency in shear-thinning viscoelastic fluids was larger than that in Newtonian fluids, regardless of viscosity. This suggests that motile sperm in cervical mucus move efficiently by means of a motion mechanism that is suited to their surrounding environment.     

Intracellular signal transduction pathways in the regulation of fowl sperm motility: Evidence for the involvement of phosphatidylinositol 3‐kinase (PI3‐K) cascade

The possible role of PI3‐K in the reversible temperature‐dependent immobilization of fowl sperm motility was investigated by using PI3‐K inhibitor (LY294002) and its inactive analogue (LY303511). The existence of the PI3‐K in fowl spermatozoa was also confirmed by Western blotting analysis. Fowl sperm motility in TES/NaCl buffer remained negligible at the avian body temperature of 40°C but was maintained vigorously when the temperature was decreased to 30°C. At 30°C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl₂ and 10 µM LY294002 or LY303511: around 70–80% of spermatozoa remained motile. In contrast, at 40°C, the motility of spermatozoa was activated immediately after the addition of Ca²⁺, but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca²⁺‐supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca²⁺ or LY303511 + Ca²⁺ were almost the same values compared with those of Ca²⁺ alone at 40°C, suggesting that the addition of LY294002 was not simply affecting membrane damage or inhibiting energy production in the spermatozoa, but may be acting on some part of the motility‐regulating cascade. Immunoblotting of sperm extract using an antibody to PI3‐K revealed a major cross‐reacting protein of 85 kDa, which corresponds to the molecular weight of the subunit of PI3‐K. These results suggest that PI3‐K may be positively involved in the calcium‐regulated maintenance of flagellar movement of fowl spermatozoa at 40°C. Mol. Reprod. Dev. 76: 603–610, 2009. © 2008 Wiley‐Liss, Inc.     

Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster

A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined. It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.     

The cause of infertility of male c-ros tyrosine kinase receptor knockout mice

Male homozygous transgenic c-ros knockout mice are sterile by natural mating, lack a part of their epididymis, and the epididymal sperm exhibit tail angulation in vivo and in vitro. To ascertain if this abnormal tail form caused the infertility, the number and nature of sperm in the tract of females mated to knockout and wild-type mice were determined. Percentage motility and numbers of sperm in the uterus 1 h after mating were similar between genotypes. The majority of the uterine sperm from the wild-type males had straight flagella, whereas 46-86% of knockout sperm were bent at the cytoplasmic droplet even when motile. Motile knockout sperm showed a 54 and 37% reduction in the straightline and curvilinear velocities compared with straight wild-type sperm. Sequential flushings of the oviduct 4 h after mating with the wild-type males contained sperm: 591 +/- 119 free, 371 +/- 70 loosely, and 122 +/- 47 tightly bound to the epithelium, but no knockout sperm were recovered from the oviduct or observed within the uterotubal junction in tissue sections. The infertility of c-ros knockout male mice can be explained by the sperm's inability to enter the oviduct, as a result of their bent tails forming the entangled sperm mass and their compromised flagellar vigor within the uterus.     

A quantitative comparison of the passage of capacitated and uncapacitated hamster spermatozoa through the uterotubal junction.

Female hamsters were artificially inseminated at the time of ovulation with an equal concentration and volume of capacitated sperm suspension in one uterus and uncapacitated sperm suspension in the contralateral uterus. When oviducts were examined 3.5-4.0 h after insemination, a significantly (paired t-test, p less than 0.05) lower number of spermatozoa were found in the oviduct from the side inseminated with capacitated sperm suspension compared to the side inseminated with uncapacitated sperm suspension. The reduction in the number of spermatozoa entering the oviduct on the side inseminated with capacitated sperm suspension was particularly evident when nearly all the spermatozoa in the suspension were hyperactivated. These results suggest that hamster spermatozoa require a progressive linear type of motility pattern to pass efficiently through the uterotubal junction and that under normal conditions in vivo, fertilizing spermatozoa initiate hyperactivated motility after entering the oviduct.     

The motility of human spermatozoa as influenced by prostasomes at various pH levels.

Human semen contains several components among which spermatozoa, membranous vesicles called 'prostasomes', secreted by the prostate gland and unorganized material. Prostasomes possess an unusual lipid composition, contain a number of proteins and small molecules and have been claimed to take a part in the immune response, in seminal fluid liquefaction and in sperm motility. Since sperm may come in contact with an acidic environment in the vagina, it may be of some interest to know whether prostasomes may affect spermatozoon motility or may protect spermatozoa upon the exposure to an acidic milieu. Human semen was supplied by donors. From whole semen we collected spermatozoa by centrifugation and used the supernatant to prepare prostasomes (centrifugation at 105,000 g for 120 min, followed by purification step on Sephadex G 200); spermatozoa were then collected by a swim-up procedure and exposed to an acidic pH medium (from 5 to 7) in the presence or absence of prostasomes. Spermatozoa motility was subsequently assessed with a superimposed image analysis system (SIAS). Results indicate that the motility of spermatozoa was affected by the pH value of the medium. Acidic media reduced the percentage of motile cells and decreased the straight line velocity of spermatozoa (VLS). Prostasomes had a protective effect and increased the percentage of motile cells. However, they did not change the characteristics of motility (curvilinear and straight). Prostasomes may be considered as a system for counteracting the negative effects of acidic pH values that may be present in the vagina after coitus.     

Evolution of the flagellar waveform of ram spermatozoa in relation to the degree of epididymal maturation.

Motility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum.     

Expression of TLR2/4 in the sperm‐storing oviduct of the Chinese soft‐shelled turtle Pelodiscus sinensis during hibernation season

The initiation of innate immunology system could play an important role in the aspect of protection for sperms long‐term storage when the sperms got into oviduct of turtles and come into contact with epithelium. The exploration of TLR2/4 distribution and expression in oviduct during hibernation could help make the storage mechanism understandable. The objective of this study was to examine the gene and protein expression profiles in Chinese soft‐shelled turtle during hibernation from November to April in the next year. The protein distribution of TLR2/4 was investigated in the magnum, isthmus, uterus, and vagina of the turtle oviduct using immunohistochemistry, and the gene expression of TLR2/4 was analyzed using quantitative real‐time PCR (qRT‐PCR). The results showed positive TLR2 protein expression primarily in the epithelium of the oviduct. TLR4 immunoreactivity was widely observed in almost every part of the oviduct, particularly in the epithelium and secretory gland membrane. Analysis of protein, mRNA expression revealed the decreased expression of TLR2/4 in the magnum compared with the isthmus, uterus, and vagina during hibernation. The protein and mRNA expression of TLR2 in the magnum, isthmus, uterus, and vagina was decreased in April compared with that in November. TLR4 protein and mRNA expression in the magnum, isthmus, uterus and vagina was decreased in November compared with that in April. These results indicated that TLR2/4 expression might protect the sperm from microbial infections. In contrast to the function of TLR2, which protects sperm during the early stages of hibernation, TLR4 might play a role in later stages of storage. The present study is the first to report the functions of TLR2/4 in reptiles.