Short-Term Storage of Rat Sperm in the Presence of Various Extenders

Abstract

Sperm preservation protocols differ among animal species because of different sperm characteristics among species. Rat sperm have extreme sensitivity to suboptimal conditions in centrifugation, pipetting and chilling due to their longer tail, the shape and size of the sperm head, and membrane composition. The aim of this study was to determine optimal conditions for short-term storage of rat sperm by evaluating their motility and membrane and acrosomal integrity in response to various extender solutions, temperatures, and durations. Motility of rat sperm was highest when stored at 22 °C; motility was 28% and 14% at 72 h in TL-HEPES and PBS extenders, respectively. The motility and membrane integrity of rat sperm fell significantly within 24 h at 4 and 37 °C. Although cold storage did not have a detrimental effect on acrosomal integrity of sperm, room temperature storage reduced acrosomal integrity after 24 h. LEY extender caused the highest loss in acrosomal integrity at 48 and 72 h. In conclusion, storage at 4 or 37 °C reduced the motility and membrane integrity of rat sperm even with short incubation periods. Rat sperm stored in TL-HEPES or PBS remained motile for at least 3 d when held at 22 °C.

Rats are used for biomedical and genomic research and provide useful models for human disease.^26,46 Assisted reproductive technologies for rats have improved markedly over the last decade. Artificial insemination (AI) is one of the assisted reproductive technologies used in rats and generally is performed to overcome logistical problems associated with natural mating. In addition, AI can be valuable when the number of sperm or proportion of motile spermatozoa is insufficient to achieve fertility through natural mating.² In addition, the use of rat models in combination with AI is an efficient tool to analyze the effects of environment toxins on male fertility.^19,25 Although offspring have been obtained through surgical AI,³³ in vitro fertilization,⁴⁴ and intracytoplasmic sperm injection³¹ using cryopreserved sperm, nonsurgical AI with frozen–thawed sperm remains unsatisfactory for intrauterine insemination because of the low quality of thawed sperm.³² However, AI using liquid stored sperm would be a beneficial option that likely accommodates sperm transport to distant laboratories and repeated insemination without a decrease in fertility.

Sperm preservation protocols vary among animal species owing to the inherent sperm characteristics of each species.^5,9 Rat sperm has extreme sensitivity to suboptimal conditions in centrifugation, pipetting, and chilling,^33,49 due to their longer tail, the shape and size of the sperm head, and membrane composition.^10,15,18 Sperm cryopreservation has achieved the production of offspring in a broad range of species.³⁶ However, acceptable levels of fertility have not been achieved in most domestic animal species,^28,36,53 and acceptable motility and fertility after thawing have not been achieved for cryopreserved rat sperm.^32,36 However, some progress in rat assisted reproductive technologies has been accomplished in regard to in vitro fertilization,⁴⁷ AI,^33,34 and intracytoplasmic sperm injection.^16,17

Long- and short-term storage of sperm has several applications in genome banking and clinical medicine.⁵ Success rates with cooled and stored sperm depend on several confounding factors including storage temperature, cooling rate, chemical composition of the extender, concentrations of reactive oxygen species, and seminal plasma composition.^5,6 When sperm is stored for more than 4 d, special extenders and high sperm concentrations are needed to compensate reduced sperm viability.¹³ The main methods for sperm storage in liquid form involve storage at room or cool temperatures. Many extenders have been developed for the short-term storage of sperm and typically include synthetic buffers combined with sugars, egg yolk, milk, glycine, and other substances.^29,40 The success of an extender rests on its ability to control of pH and osmolarity and as an energy supply.³ Tris, N-Tris(hydroxymethyl)-methylaminoethane sulfonic acid (TES), 2(N-morpholino) ethane sulfonic acid (MES), HEPES, and phosphate have similar pH buffering capacities and are used as buffers in extenders.²³ Although lactose-, TES-, Tris–citrate-, and milk-based sperm extenders are used most commonly,^6,41,54 the composition of the most optimal extender may vary according to species.¹⁸ In the current study, HEPES-buffered Tyrode lactate (TL-HEPES), Tris, modified Krebs–Ringer bicarbonate (mKRB), 8% lactose monohydrate with 20% egg yolk (LEY), and PBS were used as extenders. TL-HEPES and PBS have been reported to be suitable extenders for diluting sperm^45,49 and transporting sperm in liquid state.^24,42 mKRB has been used for rat IVF⁴⁴ and has recently been shown to positively affect the cryosurvival of rat epididymal sperm.^54,55 Tris-citrate is a well-known diluent⁴¹ that has good potential for rat sperm handling and chilling.⁵⁰

Sperm quality must be maintained during collection and transport if samples are to be used for AI or IVF. Sperm survival is much higher after short-term liquid storage than after frozen storage. For example, sperm from bulls,^7,12 stallions^4,40 and rams²⁹ can be cooled and stored at 4 °C for up to 48 h. The effects of centrifugation, physical interventions such as pipetting, various extenders, heat,^49,50 cryoprotectant, and osmotic stress⁴⁵ on rat sperm have been reported. However, optimal extenders, storage environment, and duration for storage of rat sperm have not been studied comprehensively. Therefore, the objectives of this study were to determine the effects of various extenders, holding times, and temperatures on rat sperm quality.

Materials and Methods

All chemicals were purchased from Sigma (St Louis, MO), unless otherwise stated.

Animals and sperm collection.

Outbred Sprague–Dawley male rats (age, 16 to 20 wk) were used as sperm donors. The rats were housed in accordance with the policies of the University of Missouri Animal Care and Use Committee and the Guide for the Care and Use of Laboratory Animals.²⁰ For sperm collection, male rats were euthanized by CO₂ inhalation, and cauda epididymides were excised and placed in a 35-mm culture dish containing TL-HEPES solution supplemented with 3 mg/mL BSA (fraction V). The cauda epididymides were dissected with fine scissors to allow sperm to swim out for 10 to 15 min at room temperature. The sperm suspension was drawn gently into a plastic transfer pipette (inner diameter, 2 mm; Samco, San Fernando, CA) and placed in a 5-mL tube for further experimentation. The number of spermatozoa was determined by using a Thoma hemocytometer. The final concentration of each sperm sample was about 12 to 15 × 10⁶ sperm/mL. The motility analysis was performed by using a phase-contrast microscope, and the sperm samples were held at 22 °C for further experimentation. Each experiment was performed by using sperm from a single donor and was done 6 times.

Preparation of sperm extenders.

Five extenders—TL-HEPES,⁸ Tris–citrate,⁴¹ mKRB,^48,54 LEY, and PBS (Invitrogen)—were tested. The osmolalities of the extenders were determined by using a vapor-pressure osmometer (Vapro 5520, Wescor, Logan, UT).

TL-HEPES.

TL-HEPES contained 114 mM NaCl, 3.2 mM KCl, 2 mM NaHCO_3, 0.4 mM NaH₂PO_4•H₂O, 10 mM lactic acid, 2 mM CaCl_2•2H₂O, 0.5 mM MgCl₂•6H₂O, 10 mM HEPES, 10 mL/L penicillin–streptomycin (10 mg streptomycin and 10,000 IU penicillin sulfate in 1 mL).⁸ BSA (fraction V, 3 mg/mL) was added to obtain a working solution. Phenol red was not added due to its effects to fluorescent staining. The pH and osmolality of the TL-HEPES extender were 7.2 and 290 ± 5 mOsm, respectively.

Tris.

This extender was adopted from Salamon modified Tris–citrate⁴¹ and contained 27.0 g/L Tris, 14.0 g/L citric acid, 10.0 g/L fructose, 50µg/mL streptomycin, and 75 µg/mL penicillin sulfate. The osmolality of this extender was 416 ± 5 mOsm, and the pH was adjusted to 7.0.

mKRB.

mKRB buffer contained 94.6 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl₂•0.2H₂O, 1.19 mM MgSO₄•0.7H₂O, 1.19 mM KH₂PO₄, 25.07 mM NaHCO₃, 21.58 mM sodium lactate, 0.5 mM sodium pyruvate, 5.56 mM glucose, 50 µg/mL streptomycin, and 75 µg/mL penicillin sulfate.^48,54 The pH and osmolality of the mKRB extender were 7.0 and 300 ± 5 mOsm, respectively.

LEY.

Lactose solution was prepared by dissolving 8% lactose monohydrate (w/v) in filtered, deionized water; 23 mL egg yolk was mixed with 77 mL lactose solution and centrifuged in sterile tubes at 15,000 × g for 1 h. The supernatant was transferred to a new tube, and 50 µg/mL streptomycin and 75 µg/mL penicillin sulfate were added to the solution. The pH and osmolality were 7.0 and 330 ± 5 mOsm, respectively.

PBS.

PBS extender was prepared by diluting the 10× stock and adding 50 µg/mL streptomycin and 75 µg/mL penicillin sulfate. The pH and osmolality of the PBS were 7.3 and 280 ± 5 mOsm, respectively.

Experimental design.

Sperm samples (100 µL each) in TL-HEPES were transferred into 1.5-mL centrifuge tubes and gently mixed with 900 µL of each extender to be tested. After dilution, the percentage of motile sperm was determined. Sperm samples then were transferred to 4 °C, 22 °C, and 37 °C water baths and incubated for 6, 12, 24, 32, 48, and 72 h. After incubation, sperm motility was analyzed. In addition, acrosomal integrity and the ratio of live to dead sperm were determined by using epifluorescent microscopy (Axiophot, Zeiss, Jena, Germany).

Assessment of sperm motility.

The percentage of motile spermatozoa was determined visually by direct observation and by using a phase-contrast microscope (Eclipse 600, Nikon, Tokyo, Japan) equipped with a heated (37 °C) stage. Sperm samples (10 µL each; 1 to 2 × 10⁶ spermatozoa) were placed on microscope slides (80-µm deep dual-sided chamber, 2xCell, Hamilton Thorne Biosciences, Beverly, MA), covered with coverslips, and observed at 200× magnification.

Fluorescent microscopic evaluation of plasma membrane.

Propidium iodide and SYBR14 (FertiLight, Molecular Probes, Eugene, OR) was used to determine plasma membrane integrity. Treated and control sperm samples were incubated with 5 mM propidium iodide and 1 nM SYBR14 at 37 °C for 10 min. After staining, 10 µL of sperm sample was placed on a microscope slide, covered with a coverslip, and 100 sperm were evaluated under epifluorescence microscopy (Zeiss Axiophot).

Assessment of acrosomal integrity.

Epifluorescent microscopy was used to assess acrosomal integrity after staining with Alexa Fluor-488–PNA conjugate (Molecular Probes). Treated and control samples were smeared onto microscope slides, air-dried, fixed with 99% methanol, and kept at room temperature until fluorescence staining. For staining, slides were incubated with 20 μg/mL Alexa Fluor-488–PNA at 37 °C for 30 min, washed with PBS, and analyzed by epifluorescent microscopy (Zeiss Axiophot). The images of stained sperm samples were classified into 2 groups: sperm displaying strong and moderate bright fluorescence in the acrosomal region were considered to be intact, whereas sperm displaying weak, patchy, or no fluorescence in the acrosomal region were considered to be damaged (Figure 1). We evaluated 100 sperm on each slide to determine the proportion of sperm with intact acrosomes.⁴⁹ Acrosomal integrity was not evaluated in samples in which there were no motile sperm.

Figure 1.

Representative fluorescent images of rat sperm after staining with Alexa Fluor-488–PNA. (A) Intact acrosome. (B) Damaged and partially missing acrosome.

Statistical analysis.

Statistical analysis was performed by using the GLM procedure of SAS (version 9.1, SAS Institute, 1985 Cary, NC). Experiments were done 6 times, and the data were analyzed to determine the effects of extender and time on motility and membrane and acrosome membrane integrity. Parametric data were analyzed by one-way ANOVA; when there were significant differences, the Tukey test was used for multiple comparisons. Nonparametric data were analyzed by Wilcoxon–Mann–Whitney test. Data are given as the mean ± SEM. For all statistical tests, a P value less than 0.05 indicated statistical significance.

Results

Epididymal rat sperm motility and membrane and acrosomal integrity at 0 h were similar among extenders (Figure 2). However, motility decreased to 12.5% to 28.3% after 6 h at 4 °C, and 24 h of chilling at 4 °C abolished motility and membrane integrity (Table 1). The highest motility at 4 °C and 24 h was only 9.2% (LEY extender). However, chilling did not have a detrimental effect on acrosomal integrity (Table 2), which ranged from 64.0% to 77.2% at 24 h.

Figure 2.

Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 4 °C for 6, 24, 32, 48, and 72 h. Data are presented as mean ± SEM (n = 6).

Table 1.

Membrane integrity (%) of epididymal Sprague–Dawley sperm stored at 4, 22, and 37 °C for 24, 48, and 72 h by using TL-HEPES, Tris, mKRB, LEY, and PBS extenders

Temperature	Extender	After dilution	24 h	48 h	72 h
4 °C	TL-HEPES	38.0 ± 3.2	10.0 ± 4.8a	04.0 ± 4.0	00.0 ± 0.0
	Tris	40.0 ± 3.2	05.0 ± 2.7a	00.0 ± 0.0	00.0 ± 0.0
	mKRB	34.0 ± 4.0	00.0 ± 0.0a	00.0 ± 0.0	00.0 ± 0.0
	LEY	33.0 ± 1.5	05.0 ± 1.6a	03.0 ± 3.3	00.0 ± 0.0
	PBS	32.0 ± 2.6	10.0 ± 2.3a	00.0 ± 0.0	00.0 ± 0.0
22 °C	TL-HEPES	38.0 ± 3.2	29.5 ± 5.1a	25.0 ± 4.8a	09.2 ± 3.3a
	Tris	40.0 ± 3.2	12.8 ± 3.1a	04.7 ± 3.0a	00.0 ± 0.0a
	mKRB	34.0 ± 4.0	11.8 ± 1.5a	10.3 ± 2.7a	07.8 ± 2.8a
	LEY	33.0 ± 1.5	14.2 ± 4.0a	09.8 ± 2.3a	00.7 ± 0.4a
	PBS	32.0 ± 2.6	20.5 ± 3.0a	15.0 ± 3.5a	12.0 ± 3.6a
37 °C	TL-HEPES	38.0 ± 3.2	15.0 ± 5.6a	00.0 ± 0.0	00.0 ± 0.0
	Tris	40.0 ± 3.2	13.0 ± 5.5a	00.0 ± 0.0	00.0 ± 0.0
	mKRB	34.0 ± 4.0	02.0 ± 2.0a	00.0 ± 0.0	00.0 ± 0.0
	LEY	33.0 ± 1.5	00.0 ± 0.0a	00.0 ± 0.0	00.0 ± 0.0
	PBS	32.0 ± 2.6	09.0 ± 4.0a	00.0 ± 0.0	00.0 ± 0.0

Data are presented as mean ± SEM (n = 6).

^aValues within a temperature and duration are significantly (P < 0.05) different.

Table 2.

Acrosomal integrity (%) of Sprague–Dawley epididymal rat sperm stored at 4, 22, and 37 °C for 24, 48, and 72 h

Temperature	Extender	After dilution	24 h	48 h	72 h
4 °C	TL-HEPES	91.8 ± 2.7	74.7 ± 4.6	71.8 ± 2.6	not done
	Tris	91.8 ± 1.9	69.7 ± 5.6	not done	not done
	mKRB	93.3 ± 2.2	not done	not done	not done
	LEY	91.8 ± 3.5	77.2 ± 4.3	73.7 ± 3.4	not done
	PBS	90.2 ± 2.2	64.0 ± 6.6	not done	not done
22 °C	TL-HEPES	91.8 ± 2.7	78.7 ± 6.1	67.5 ± 6.6a	62.2 ± 7.6a
	Tris	91.8 ± 1.9	77.7 ± 4.4	75.7 ± 1.9a	not done
	mKRB	93.3 ± 2.2	74.0 ± 5.6	71.8 ± 3.9a	67.8 ± 3.7a
	LEY	91.8 ± 3.5	75.7 ± 5.4	54.5 ± 7.9a	37.0 ± 2.8a
	PBS	90.2 ± 2.2	75.5 ± 5.8	67.7 ± 9.0a	57.3 ± 8.9a
37 °C	TL-HEPES	91.8 ± 2.7	74.0 ± 2.6	not done	not done
	Tris	91.8 ± 1.9	71.2 ± 3.6	not done	not done
	mKRB	93.3 ± 2.2	72.0 ± 1.9	not done	not done
	LEY	91.8 ± 3.5	not done	not done	not done
	PBS	90.2 ± 2.2	78.2 ± 3.7	not done	not done

Data are presented as mean ± SEM (n = 6).

^aValues within a temperature and duration are significantly (P < 0.05) different.

After 72 h of storage, motility and membrane integrity were significantly (P < 0.05) higher at 22 °C compared with 4 and 37 °C. Sperm motility at 6 h was significantly (P < 0.05) affected by the extender (Figure 3). Sperm preserved in Tris showed the greatest decline in motility (7.0% at 24 h), whereas sperm in TL-HEPES and PBS retained the highest (P < 0.05) motility (28.0% and 14.2%, respectively) and membrane integrity (9.2% and 12.0%, respectively) at 72 h. Sperm motility and membrane integrity declined gradually from 0 to 72 h during storage at room temperature (that is, 22 °C). Acrosomal integrity was not significantly affected by extender type, except at room temperature for 48 and 72 h. Acrosomal integrity was 67.5% and 62.2% after 48 and 72 h of storage, respectively, in TL-HEPES at 22 °C.

Figure 3.

Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 22 °C for 6, 24, 32, 48, and 72 h. Data are presented as mean ± SEM (n = 6). *, Values within the same group are significantly (P < 0.05) different from each other.

Storage at both 4 and 37 °C caused loss of motility and membrane integrity, even with shorter storage periods. After 6 h at 37 °C, motility (Figure 4) and membrane integrity (Table 1) parameters were decreased significantly. After 24 h at 37 °C, motility and membrane integrity were negligible for all extenders except TL-HEPES. Sperm motility in TL-HEPES was 15.0% after 24 h but was abolished at 32 h.

Figure 4.

Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 37 °C for 24, 48, and 72 h. Data are presented as mean ± SEM (n = 6). *, Values within the same group are significantly (P < 0.05) different from each other.

Discussion

Two main factors influence sperm cell function after ejaculation and during in vitro storage: the temperature at which the sperm is stored after dilution, and the composition of the suspension medium.²¹ Although sperm from bulls,^7,12 stallions^4,40 and rams²⁹ can successfully be kept at 4 to 5 °C, boar sperm stored at ambient temperature performed better.²¹ Boar and rat^21,35,49 sperm are very susceptible to cold shock. To successfully store rat sperm, species-specific storage conditions and extenders should be defined. To that end, we tested different storage extenders, temperatures, and durations for rat sperm.

Sperm storage temperatures play an important role in extending the lifespan and functionality of sperm. Storage at cold temperature reduces the metabolic activity of sperm, but not all changes associated with lower temperatures are beneficial to sperm.⁴ Our study showed that rat sperm can be kept at room temperature (that is, 22 °C) for as long as 72 h without dramatic loss of motility. Among the 5 extenders tested, sperm motility at 22 °C decreased to 49.2% to 56.7% after dilution (0 h) to 14.2% to 28.0% at 6 h, and after storage for 24 h at 4 °C, motility had decreased to 9.2%, 4.2%, and 3.3% in LEY, PBS, and TL-HEPES, respectively. Similarly, after 24 h of sperm storage at 4 °C, the membrane integrity of sperm was reduced to 5.0%, 10.2%, and 9.8% in LEY, PBS, and TL-HEPES, respectively. In one study, chilling for 30 min and rewarming to 37 °C caused 30% to 40% motility loss.⁴⁹ In the current study, sperm motility and membrane integrity were affected detrimentally by even brief storage in cold. Our results also showed that sperm storage at 4 °C greatly reduced motility and membrane integrity compared with those after storage at 22 °C. These data suggest that the extenders we tested were not particularly effective in protecting rat sperm from cold shock; although egg yolk supported sperm integrity, it lacked a sufficient hypothermic protective effect on rat sperm.

For most species, the optimal liquid sperm storage temperature is 4 to 5 °C,^{4,7,12,29,38,40,51} but not for rats and boars.²¹ For these species, sperm storage at room temperature is an alternative. During the storage of sperm at room temperature, diluting media extends the survival of sperm by decreasing its metabolic activity.¹¹ To inhibit metabolic activity and maintain the viability of sperm, a suitable extender is required.^31,52 For this purpose, several extenders have been developed to decrease the metabolic activity of sperm at ambient temperature (15 to 18 °C).²¹ The main changes that occur during liquid storage of sperm include reduction in the motility and morphologic integrity of spermatozoa. These changes may result from the accumulation of toxic metabolic products (mainly reactive oxygen species),^30,41 sperm ageing processes,²¹ and increased pH.⁵² The high metabolic activity of sperm is maintained for only a limited time period¹³ due to the production of additional toxic products.^41,43 In addition, an increase or decrease in the pH of the extender during liquid storage at room temperature was significantly correlated with decreased motility.^22,52 In the current study, sperm stored in PBS and LEY maintained higher motility even though Tris and TL-HEPES have greater buffering capacity. In agreement with our study, it has been reported that buffering capacity alone is sufficient for the maintenance of sperm viability.³¹ In our study, sperm extended in TL-HEPES and PBS and stored at 22 °C maintained 28.0% and 14.2% motility, respectively, at 72 h. Tris extender has been used widely to cryopreserve sperm from other species;⁴¹ however, after 24 h of storage in Tris extender, the motility of rat sperm was reduced to 6.7% (from 52.0%), and membrane integrity dropped to 12.8% (from 40.0%) in our study. In accordance with the present study, the storage of boar, mouse, and rat sperm was more effective previously at ambient than cold temperature.^21,43 In our study, cold storage did not have a detrimental effect on the acrosomal integrity of sperm, but acrosomal integrity declined gradually after 24 h at room temperature. For example, acrosomal integrity diminished from 91.8% to 37.0% in LEY extender (P < 0.05). The decline in acrosomal integrity may be due to an extender effect. Similarly, skim milk extender shortens the lifespan of equine sperm, due to acrosomal effects.³⁹

Sugars are used in extenders as cryoprotectants and sources of energy.^1,27,41 Spermatozoa can produce the energy needed to maintain cell metabolism by using the glucose in extenders or seminal plasma.¹³ In our study, TL-HEPES and PBS resulted in significantly better survival of sperm. In contrast, LEY, Tris, and mKRB—which contain higher sugar concentrations—significantly decreased sperm motility. Our current results suggest that excess sugar in the diluent decreases the lifespan of sperm, probably secondary to an increase in metabolic activity. Therefore, to prevent increases sperm metabolism during liquid state storage in room temperatures, low concentrations of sugars may be important for sperm survival. In the ejaculate, metabolic activity of sperm is very high and can be maintained over a limited period only. Our study showed that sperm storage at 37 °C led to reduced sperm motility and membrane integrity in a short time of period. Sperm only retained 15.0% and 5.0% motility in TL-HEPES and PBS, respectively, at 24 h. The decline in sperm survival in body temperature can be attributed to the increased energy consumption and rate of metabolism that spermatozoa manifest at warmer temperatures (35 °C).¹³

Sperm motility analysis provides a simple estimate of sperm quality but gives very little information on fertilizing capacity. Cell viability, acrosomal integrity, and mitochondrial function provide more accurate descriptions of the fertilization capacity of spermatozoa.¹⁴ For this reason, all sperm parameters should be taken into consideration to evaluate sperm fertilization capability. At 22 °C storage, acrosomal integrity decreased from 91.8% to 62.2% in TL-HEPES at 72 h whereas motility and membrane integrity decreased from 57.7% to 28.0% and from 38.0% to 9.2%, respectively. In our study, acrosomal integrity was the parameter least affected by liquid storage. Similarly, acrosomal integrity was maintained during liquid storage of sperm from rams³⁸ and boars.³⁷ Interestingly, values for membrane integrity were lower than those for motility in the current study. Other authors have obtained similar results and reported low membrane integrity (18%) compared with motility (72%) in native sperm. In addition, the authors reported a 69.2% pregnancy rate when cryopreserved rat sperm with low motility (9.0%) was used.³³ This previous and our current results indicate that the liquid storage method we tested here can successfully be used for AI in rats.

In summary, storage of rat sperm in the cold or at body temperature decreased its motility and membrane integrity even during short incubation periods. Sperm extended in LEY or TL-HEPES was somewhat protected against chilling injury but did not retain motility at 24 h. Although lactate and Tris have been used widely to cryopreserve sperm from other species including mice, they were suboptimal against chilling injury for rat sperm. In the current study, the TL-HEPES and PBS diluents enabled us to preserve rat sperm in vitro for 3 d.