Microtubule and chromatin organization during the first cell-cycle following intracytoplasmic injection of round spermatid into porcine oocytes

The objective of this study was to determine microtubule assembly and chromatin configuration in porcine oocytes during the first cell cycle following round spermatid injection into matured porcine oocytes in the presence or absence of electrical stimulation. The oocytes with two large pronuclei and two polar bodies were classified as normal fertilization at 6 to 8 h following injection. The incidence of normal fertilization following round spermatid injection with electrical stimulation was significantly higher (21/45, 47%) than that following injection alone (6/39, 15%). Although a small microtubular aster was organized near the decondensed spermatid chromatin in some oocytes (2/6, 33%, spermatid injection alone; 9/21, 29%, spermatid injection and electrical stimulation), it did not enlarge nor fill the cytoplasm. Instead, a dense network of microtubules in the cytoplasm was organized from cortex. At 12 to 15 h after injection, we classified the oocytes with closely apposed pronuclei as normal fertilization. The electrical stimulation following spermatid injection enhanced (P < 0.05) the incidence of normal fertilization (18/54, 33%) compared with spermatid injection alone (7/52, 13%). During pronuclear movement, the maternally derived microtubules filled the whole cytoplasm, which appeared to move male and female chromatin. Mitosis and two-cell division were observed at 20 to 24 h after spermatid injection with electrical stimulation (12/41, 29%). At mitotic metaphase, the microtubular spindle had focused astral poles, and chromosomes were aligned on the spindle equator. During mitosis, asters were assembled at each spindle pole, and they filled the cytoplasm. These results suggested that round spermatid nuclei of the pig can develop into a morphologically normal pronucleus in matured porcine oocytes and are competent to participate in syngamy with the ootid chromatin. In addition, functional microtubules for complete fertilization with spermatid were not associated with male-derived centrosome but were organized solely from maternal stores. Mol. Reprod. Dev. 50:221–228, 1998. © 1998 Wiley-Liss, Inc.     

Differential development of rabbit embryos following microinsemination with sperm and spermatids

Microinsemination is the technique of delivering male germ cells directly into oocytes. The efficiency of fertilization after microinsemination and subsequent embryo development may vary with the animal species and male germ cells used. The present study was undertaken to observe the in vitro and in vivo developmental ability of rabbit embryos following microinsemination with male germ cells at different stages. First, we assessed their oocyte-activating capacity by injecting them into mouse and rabbit oocytes. The majority of mouse oocytes were activated irrespective of the type of rabbit male germ cell injected (61-77%), whereas rabbit oocytes were activated differently according to the type of male germ cells (89%, 75%, and 29% were activated by spermatozoa, elongated spermatids, and round spermatids, respectively; P < 0.05). After 120 hr in culture, 66%, 45%, and 13%, respectively, of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts (P < 0.05). Additional electric pulse stimulation of round spermatid-injected oocytes increased the blastocyst rate to 43%. After 24 hr in culture, some four to eight cell embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from spermatozoa and elongated spermatids, but not from round spermatids. Karyotypic analysis at the morula/blastocyst stage revealed that the majority of round spermatid-derived embryos had abnormal ploidy (8 out of 12 embryos). Our study indicates that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. As these differential developmental patterns are similar to those reported for humans in vitro and in vivo, rabbits may provide an alternative small animal model for studying the biological nature and molecular basis of human microinsemination techniques, especially those using immature male germ cells.     

Centrosomal function assessment in human sperm using heterologous ICSI with rabbit eggs: a new male factor infertility assay

Sperm centrosomal function was assessed by immunocytochemical analysis after the injection of human sperm into mature rabbit eggs. Three hours after intracytoplasmic sperm injection (ICSI), an astral microtubule array from the base of the human sperm was observed in the rabbit eggs. This sperm aster expanded in the egg cytoplasm, concomitant with pronuclear formation, and a dense microtubule array was organized at the time of pronuclear centration. Using fertile donor sperm, the sperm aster formation rate at 3 hr after ICSI was 35.0 +/- 1.5%. Using sperm from infertile patients, the average aster formation rate was lower (25.4 +/- 14.8%, P<0.05). Among infertile cases, there was no correlation between sperm aster formation rates and conventional parameters of semen analysis. However, the sperm aster formation rate correlated with the embryonic cleavage rate following human in vitro fertilization (IVF). These data suggest that this assay reflects sperm function during embryonic development after sperm entry and that reproductive success during the first cell cycle requires a functional sperm centrosome. Furthermore, sperm centrosomal function cannot be predicted from conventional parameters of semen analysis. We propose that insufficient centrosomal function could be the cause of certain cases of idiopathic infertility. These assays may lead to the discovery of new types of infertility, which have previously been treated as "unexplained infertility," and may also lead to the treatment of infertility incurable even by ICSI. Consequently, an accurate and relevant assay to help assure couples of the success of fertilization is warranted, perhaps prior to ICSI therapy.     

Development of normal mice after microinjection of round spermatids into oocytes stored at room temperature for one day

Early studies have shown that some mouse cumulus-oocyte complexes (COCs) stored at room temperature for 24 hr still retained full developmental potential. In this study, we stored mouse COCs and denuded oocytes (DOs) at room temperature for 24 hr and activated these oocytes with 10 mM SrCl(2) or injected the oocytes with round spermatids. We found that DOs were better than COCs when stored at room temperature for 1 day and more normal oocytes were obtained when COCs were stored in more H-CZB medium at room temperature for 1 day. The rates of normal oocytes were significantly different after preservation with three schemes (90.01%, 55.81%, and 86.70%, P < 0.05). Our results also indicated that oocytes stored at room temperature for 1 day were fertilized normally (extrusion of the second polar body and formation of male and female pronuclei [PN]) after microinjection of round spermatid nuclei, and that the existence of cumulus cells (CCs) during oocyte storage did not significantly influence the early cleavage but had a detrimental effect on later embryo development and full-term development. After fertilization, most embryos developed to two-cell stage after being cultured for 24 hr, and the development rates of four- to eight-cell embryos between two experiments were similar. However, the rates of morula/blastocyst formation were significantly different (47.44% and 26.27%, respectively, P < 0.05). The birth of four healthy pups from stored DOs indicated that the storage of DOs at room temperature for 1 day might become a practical procedure in mammalian reproduction.     

Functional role of centrosomes in spindle assembly and organization

The centrosome is the main MT organizing center in animal cells, and has traditionally been regarded as essential for organization of the bipolar spindle that facilitates chromosome segregation during mitosis. Centrosomes are associated with the poles of the mitotic spindle, and several cell types require these organelles for spindle formation. However, most plant cells and some female meiotic systems get along without this organelle, and centrosome‐independent spindle assembly has now been identified within some centrosome containing cells. How can such observations, which point to mutually incompatible conclusions regarding the requirement of centrosomes in spindle formation, be interpreted? With emphasis on the functional role of centrosomes, this article summarizes the current models of spindle formation, and outlines how observations obtained from spindle assembly assays in vitro may reconcile conflicting opinions about the mechanism of spindle assembly. It is further described how Drosophila mutants are used to address the functional interrelationships between individual centrosomal proteins and spindle formation in vivo. © 2004 Wiley‐Liss, Inc.     

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and fi0 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of thedonor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES ceils maintainthe capability of sustained growth in an undifferen tiated state, and form embryoid bodies, which, on furtherinduction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that expressmarkers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NTto rabbit eggs retain phenotypes similar to those of conventional human ES ceils, including the ability toundergo multilineage cellular differentiation.     

Active demethylation of individual genes in intracytoplasmic sperm injection rabbit embryos

Intracytoplasmic sperm injection (ICSI), as an assisted reproduction technique, has been widely used in animal and human. However, its possible effect on epigenetic changes has not been well studied. To investigate whether ICSI can induce aberrant DNA methylation changes in rabbit preimplantation embryos, we examined the methylation status of the SP-A promoter region and the satellite sequence Rsat IIE by bisulfite-sequencing technology. The SP-A promoter region was extensively demethylated before the first round of DNA replication commences, and the unmethylated status was maintained until morula when dynamic remethylation occurred. A similar but more moderate demethylation process was observed in satellite sequence Rsat IIE. These results are in contrast with the previous reports of no active demethylation in normal rabbit embryos, suggesting that the active demethylation we observed may be induced by ICSI.     

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.     

Production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm

As a method of producing transgenic animals, spermatozoa have been used to fertilize mammalian oocytes through natural copulation, artificial insemination (AI), and in vitro fertilization (IVF). Our objective was to produce live piglets expressing the enhanced green fluorescent protein (eGFP) by the modified ICSI procedure based on Yong et al. (2003) (Hum. Reprod. 18:2390) where this procedure resulted in an improvement in development in vitro as compared to conventional ICSI and IVF. After injecting frozen-thawed sperm, recovered from the descendant of a transgenic boar derived by oocyte transduction, into in vitro matured oocytes the injected oocytes were surgically transferred into the oviduct of six surrogate gilts. Two gilts (33%) became pregnant. One gave birth to a healthy male piglet. Expression of the eGFP was easily observed in the nose and hooves by direct epifluorescent examination in the newborn piglet. These results show the production of the first viable transgenic piglet by in vitro maturation and our new sperm injection method.     

Phosphatidylcholine and phosphatidylinositol-specific phospholipases C of bull and rabbit spermatozoa.

Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine-specific phospholipase C (PC-PLC) that hydrolyzes L-alpha-dipalmitoyl-(choline-methyl-14C-153.0 Ci/mmol and a phosphatidylinositol-specific phospholipase C (PI-PLC) that hydrolyzes L-alpha-(Myo-Inositol-2-3H (N)-5.2 Ci mmol. PI-PLC from bull sperm acrosome has been purified 568 x fold with a specific activity 6.25 +/- 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC-PLC remained unaffected by varying concentrations of Ca2+, whereas PI-PLC activity was significantly increased. The bulk of PI-PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC-PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell.     

Human sperm injection into the perivitelline space (SI-PVS) of hamster oocytes: Effect of sperm pretreatment by calcium-ionophore A23187 and freezing-thawing on the penetration rate and polyspermy

Calcium-ionophore A23187 and freezing-thawing were used as sperm treatments before human sperm injection into the perivitelline space (SI-PVS) of hamster oocytes. The penetration rate (PR) was higher when SI-PVS was performed with calcium-ionophore-treated (28%) or frozen-thawed (51%) sperm than with untreated sperm (8%). Optimal PR occurred when five calcium-ionophore-treated (57%) or frozen-thawed (71%) sperm were injected under the zona pellucida. When the sperm:egg ratio was 1:1, PR was higher for calcium-ionophore-treated (18.5%) or frozen-thawed (27.8%) sperm than for untreated sperm (0.0%). Calcium-ionophore sperm treatment had no effect on the polyspermic oocyte rate (POR) or the mean number of swollen sperm nuclei per penetrated oocyte (Pd) or per injected sperm (SR). This may result from premature oocyte activation induced by Ca-ionophore. However, POR was higher with frozen-thawed (74%) than with untreated (50%) or Ca-ionophore-treated (50%) sperm. Whatever the sperm treatment, there was a trend toward a lower SR as the number of injected sperm increased. Cytoplasmic regulation of polyspermy in the hamster oocyte is discussed.     

X-ray induction of dominant lethals in late spermatids and mature spermatozoa of Drosophila melanogaster: the role of oxygenation

The role of oxygenation in determining the sensitivity to the induction of dominant lethals was examined in late spermatids and mature spermatozoa of Drosophila melanogaster. 0–2-h-old or 7-day-old Oregon-K males were irradiated with different X-ray exposures in nitrogen, air or in oxygen and the frequencies of dominant lethals induced in these stages were studied. The results obtained confirm and extend Sobels' earlier observations and the interpretation derived therefrom namely, that under normal conditions in air, mature spermatozoa are characterised by a higher degree of oxygenation than late spermatids and this difference is sufficient to explain the differential response of these stages. Similar Oxygen-Enhancement-Ratios(OERs) (of about 2) were obtained for both the cell stages. The present data also revealed that late spermatids are significantly less sensitive than mature spermatozoa to the X-ray-induction of dominant lethals in a nitrogen atmosphere. A plausible mechanism is suggested to explain this observation.     

Flow sorting of X and Y chromosome-bearing mammalian sperm: Activation and pronuclear development of sorted bull,boar, and ram sperm microinjected into hamster oocytes

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio-threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P <.05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P <.05). Treatment of sperm with DTT increased the activation rate (P < .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome-bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.     

Different occurrence rates of centrally located cytoplasmic granulation in one cohort oocytes show distinctive embryo competence and clinical outcomes

Centrally located cytoplasmic granulation (central granulation) is a common cytoplasmic dysmorphism in human oocytes retrieved after controlled ovarian hyperstimulation (COH). In order to achieve a better understanding of its formation and effects on clinical outcomes, we retrospectively analyzed 422 ICSI treatment cycles. Three groups of patients were classified according to the ratio of central granulation occurrence in one egg cohort, as partial granulation, all granulation and control groups. The partial granulation group had a significantly lower BMI and higher AMH level compared to the control or all granulation groups. Consistent with these distinctive features in the partial granulation group, fertilization and blastocyst formation rates were reduced significantly in the partial granulation group but not in the all granulation group. Furthermore, the clinical outcomes in fresh embryo transfer cycles were dramatically reduced in the partial granulation group compared with the control group. However, in FET cycles, all three clinical outcomes were significantly reduced in the all granulation group but not in the partial granulation group. We propose that partial granulation may reflect a specific population of patients, and that the central granulation structure is sensitive to cryopreservation.     

Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig

The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 °C for up to 48 h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Cζ (PLCζ) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCζ in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18 h at 24 °C (78%, 62%, and 35%, respectively) and for 48 h at 14 °C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCζ in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.     

Initiation,prolongation, and reactivation of the motility of salmonid spermatozoa

The motility of salmonid spermatozoa initiated by dilution of the milt with ovarian fluid or isotonic saline is brief duration; it was believed that it can be activated only once in the life of the spermatozoon. Dilution of the milt with an equal volume of isotonic saline (0.12 M-NaCl) containing 5 mM-3-isobutyl-1-methylxanthine (MIX) prolonged and intensified sperm motiliy. When motility had stopped after initial mobilization with saline or ovarian fluid, it could be reactivated by addition of MIX; reactivated spermatozoa fertilized eggs. Dilution with saline containing K⁺ (24 mEq/liter) did not initiate sperm motility even in the presence of MIX. The spermatozoa were mobilized by subsequent with 0.12 M-NaCl. The concentration of adenosine triphosphate (ATP) in sperm suspensions dropped on dilution with saline and rose as motility ceased, but declined without subsequent recovery following dilution with MIX-saline. The concentration of cyclic adenosine monophosphate (cAMP) rose and fell sharply on initiation of motility and rose again after motility had declined. While salmonid spermatozoa can be mobilized by dilition with saline alone, the effectiveness of MIX in reactivating “spent” spermatozoa supports the assumption that cAMP plays a role in the initiation of sperm motility.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

Structural and Functional Analyses of the FAM46C/Plk4 Complex

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Addition of butylated hydroxytoluene (BHT) in tris-based extender improves post-thaw quality and motion dynamics of dog spermatozoa

The aim of this study was to evaluate the interaction of different concentrations of butylated hydroxytoluene (BHT) in a tris-based extender on semen quality parameters in post-thawed dog semen. Twenty-four ejaculates were collected from eight male Beagle dogs using an artificial vagina. Pooled semen was diluted with a tris-based extender supplemented with 0 (control), 0.5, 1.0, 1.5, and 2.0 mM BHT, at a final concentration of 200 × 10⁶ spermatozoa/mL. After thawing, sperm samples were assessed for motility parameters (CASA), membrane integrity (SYBR-14/PI), acrosome integrity (FITC-PNA), mitochondrial activity (JC-1/PI), malondialdehyde (MDA) concentration, and glutathione peroxidase (GPx) activity. The total motility, progressive motility, and average path velocity of the frozen-thawed sperm were significantly higher in the BHT1.5 group than in the control and the other sample groups (P < 0.05). Higher values of straight-line velocity, curvilinear velocity, amplitude of the lateral head displacement, and linearity were observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (P < 0.05). The BHT1.0 and BHT1.5 groups had higher percentages of straightness and acrosome integrity than the other groups (P < 0.05). Beat cross frequency, plasma membrane integrity, and GPx activity of the BHT1.5 and BHT2.0 groups were higher than those of the control (P < 0.05). A lower concentration of MDA was observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (BHT0) (P < 0.05).Our results indicate that 1.5 mM BHT is the optimal concentration for improving the post-thaw quality of canine spermatozoa.