Effect of season on the dynamics of cat sperm DNA fragmentation.

BACKGROUND: Feline species undergo reproductive seasonality; thus, sperm characteristics, such as DNA integrity, can be affected by the photoperiod. This study was conducted to determine the effect of seasonal changes on sperm quality and on the dynamics of sperm DNA fragmentation. Epididymal spermatozoa were collected from 36 tomcats subjected to bilateral orchiectomy during breeding (BS) and non-breeding (NBS) seasons. Sperm samples were obtained by cutting the cauda epididymis and assessed for sperm motility, concentration, acrosome integrity, plasma membrane integrity and sperm morphology. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test after 0, 6, and 24 h of incubation at 37 °C. RESULTS: The total sperm motility and plasma membrane integrity values were greater during the BS, while the percentages of abnormal sperm and head defects were lesser (p < 0.05). No significant differences in DNA fragmentation were found between seasons after sperm collection. DNA damage was greater after 24 h of incubation at 37 °C in both seasons, although the percentage of spermatozoa with fragmented DNA was significantly lesser in the BS than in the NBS at 24 h (p < 0.05). CONCLUSIONS: The study suggests seasonal changes in some of the quality parameters of cat sperm. DNA fragmentation dynamics were affected by the time of incubation and reproductive season; therefore, this technique might be used as an additional tool to test the potential fertility of semen samples used in feline-assisted reproduction.

Development of a laparoscopic technique for inguinal hernioplasty in standing horses.

BACKGROUND: Most previously described techniques for laparoscopic inguinal hernioplasty (IH) in horses require advanced laparoscopic skills. Our objective was to describe a new laparoscopic IH technique using a surgical anchoring system. METHODS: Standing laparoscopic IH was performed unilaterally in eight experimental stallions, using the contralateral inguinal canal (IC) as a control. A polyether ether ketone harpoon was anchored in the craniolateral aspect of the vaginal ring, and an extracorporeal knot was used to fix the device. Clinical evaluation, including testicular palpation and lameness examination, was conducted before and for 4 weeks after surgery. Repeat laparoscopy was performed 28 days later. RESULTS: Standing laparoscopic IH was performed in all horses with a surgical time of 38 ± 12.85 minutes. In two animals, a small peritoneal tear occurred that did not require repair. No other complications were recorded. On repeat laparoscopy, all devices were in place, and the IC remained partially closed in all horses. LIMITATIONS: The procedure was performed on normal experimental horses and has not been employed on horses that have had an inguinal hernia. CONCLUSIONS: This new standing laparoscopic hernioplasty technique provides another potential method for simple partial closure of the IC in stallions at risk of or with history of inguinal herniation.

Specific phosphodiesterase type-10 inhibitor, papaverine, added after the cooling period improves canine sperm quality.

The use of chilled semen has gained increasing interest in canine reproductive services. The addition of phosphodiesterase (PDE) inhibitors that increase the intracellular cyclic adenosine monophosphate levels may improve sperm motility. The purpose of this study was to examine the quality of sperm under the effect of the specific PDE-10 inhibitor (papaverine) added after storage for 1, 2, and 3 days at 5 °C. The ejaculates were obtained from 5 healthy Beagle dogs by digital manipulation. After collection, ejaculates were pooled, extended and cooled at 5 °C during 3 days. Sperm parameters were tested 30 min after the addition of different papaverine (PA) concentrations: 0, 5, 10 and 20 µM. Sperm motility (CASA), viability (PI/FITC-PNA) and capacitation status (chlortetracycline assay) were evaluated. The results showed that the addition of PA has no effect on sperm samples at day 0. However, concentrations of 5 and 10 µM increased (p < .05) sperm motility kinetics and viability significantly compared to the control at day 1, day 2 and day 3 of cooling. The addition of 20 µM PA decreased (p < .05) sperm quality parameters significantly and increased the percentage of capacitated/acrosome-reacted spermatozoa. In conclusion, the addition of 5 and 10 µM PA concentrations after cooled storage improved canine sperm quality.

Morphological Characteristics of the Sperm of the Peregrine Falcon (Falco peregrinus) during the Reproductive Season.

The morphological characteristics of different sperm cells (normal, abnormal, and immature) in the peregrine falcon during the reproductive season were analysed. We also classified the main sperm defects found in semen. Semen samples were collected from mature peregrine falcons via cloacal massage and stained with Diff-Quik stain. The percentages of normal, abnormal, and immature sperm cells were determined by bright-field optical microscopy. The number of normal spermatozoa were greater at the initial stage and subsequently decreased during the middle and later stages of the reproductive season (p < 0.01). In contrast, the percentage of abnormal spermatozoa increased significantly in the middle and end stages of the reproductive season (p < 0.05), whereas the proportion of immature spermatozoa remained stable during the study. Head defects represented the greatest proportion of morphological abnormalities, followed by the defects in the tail and midpiece regions. A small percentage of multiple defects and cytoplasmic droplets were also observed in the falcon spermatozoa. The findings of this study might be important for the development of future conservation protocols for falcon sperm.

Correlation of Prostatic Artery Blood Flow Assessed by Doppler Ultrasonography with Semen Characteristics in Beagle Dogs.

Pulsed-wave Doppler ultrasonography (PwD) is a method used to rapidly and noninvasively assess blood flow dynamics of the canine prostate. Modifications in gland vascularization can affect seminal plasma production and consequently sperm quality. The aim of this study was to determine the normal blood flow parameters of the prostate artery in beagle dogs and to analyze the correlations between vascular flow and semen quality characteristics. PwD was performed on five beagle dogs (5-6 years) measuring vascular features in four different locations of the prostatic artery (cranial, subcapsular, parenchymal and caudal); the measured features were peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI) and pulsatility index (PI). Ejaculates were obtained using digital manipulation and semen quality was evaluated by determining macroscopic (total volume, sperm-rich fraction volume, color and pH) and microscopic (sperm motility, morphology, viability and acrosome integrity) characteristics. The values of PSV, PI and RI in cranial and caudal prostatic arteries were significantly higher than in subcapsular and parenchymal arteries (p < 0.05). Moreover, a positive correlation of PSV value in the cranial region of the prostatic artery with total ejaculate volume (p < 0.01, r = 0.612) and sperm concentration (p < 0.01, r = 0.587) was determined. PI index was negatively correlated with sperm concentration (p < 0.01, r = -0.709). In conclusion, the results suggest that the prostatic artery blood flow parameters can affect macroscopic semen quality characteristics in healthy dogs.

Colloid centrifugation reduces bacterial load in chilled dog semen.

Conventional semen extenders contain antibiotics to prevent bacterial growth. Finding alternatives would be beneficial to minimize the development of bacterial resistance mechanisms. The aim of this study was to determine the effect of Single Layer Centrifugation (SLC) with Canicoll of dog semen on microbial load and sperm quality during cooled storage. Twenty-four ejaculates were obtained from healthy dogs by digital manipulation. Samples were diluted in Tris-citrate-fructose extender without antibiotics and divided into two treatment groups: SLC-selected samples and unselected samples. Sperm motility (CASA), viability and acrosome integrity (PI/FITC-PNA) as well as bacterial load of each microorganism species (colony-forming units/mL) were assessed at 0 and 48 h of storage at 4 °C. Results indicate SLC-selected dog spermatozoa have greater percentages of motility, viability and acrosome integrity (P < 0.05). Bacterial growth in SLC sperm samples was less (P < 0.05) than unselected samples. Removal of individual bacterial species varied from 91 % to 98 % for Escherichia coli (91.62 %), Streptococcus spp. (98.18 %), Staphylococcus spp.(95.33 %) and Pseudomonas spp. (92.50 %). In conclusion, the use of SLC with Canicoll has the potential to decrease bacterial load in chilled dog semen.

Sperm Tail Defects and Abnormal Testicular Blood Flow in a Beagle Dog: A Case Report.

A 5-year-old male Beagle dog produced ejaculates with a high percentage of spermatozoa with abnormal morphology, especially sperm tail defects. Although libido and semen volume were normal, ejaculates showed asthenospermia, oligozoospermia, and teratozoospermia. The spermatozoa exhibited morphologic defects affecting the flagellum, mainly coiled tails with or without macrocephalia (33.5 ± 2.1%), bent tails (18.3 ± 3.4%), and proximal cytoplasmic droplets (6.7 ± 2.8%). The peripheral plasma testosterone level was 2.76 ± 0.21 ng/mL. The resistive index and the pulsatility index from marginal and intratesticular vessels measured by Doppler ultrasound showed higher values in the right testicle than in the left testicle. Histologic evaluation revealed focal reduction in the number of germ cells and sperm in the seminiferous tubules in the right testicle. This is the first report that describes simultaneously the presence of sperm tail defects in the ejaculate and changes in the blood flow of testicular vessels in the dog.

Alternatives in Donkey semen cryopreservation: Mare vs. Jenny Colostrum.

The aim of this study was to test and compare two new components in extenders for freezing donkey semen: mare colostrum and jenny colostrum. Colostrum was obtained from four mares and four jennies right after the foal's birth. Ejaculates were collected from five fertile donkeys. Sperm samples were pooled, diluted and cryopreserved in three different experimental extender groups: lactose supplemented with egg yolk extender (20%) as the control group, lactose supplemented with jenny colostrum extender (20%), and lactose supplemented with mare colostrum extender (20%). After thawing, we evaluated the sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional by HOS test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. The results demonstrated that lactose-jenny colostrum extender displayed significantly higher values (p < .05) in nearly all parameters evaluated - Total Motility, Viability, HOS test, VCL, VSL, VAP, LIN, STR and WOB -, compared with mare colostrum and egg yolk extenders after thawing. In conclusion, the extender containing jenny colostrum used for donkey semen cryopreservation improved the donkey sperm quality after the freezing-thawing process.

Effect of Mare Colostrum in Extenders for Freezing Stallion Semen.

This study aimed to evaluate the addition of mare colostrum in stallion freezing extenders to improve sperm quality. First, colostrum samples were collected from four mares after the foal's birth and their composition was determined. Ejaculates were collected from nine fertile stallions. Sperm samples were pooled, diluted, and cryopreserved into three experimental extender groups: Lactose-based extender supplemented with mare colostrum (20%), lactose-based extender supplemented with egg yolk (20%), and BotuCrio. The quality of the post-thaw semen samples were evaluated assessing sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodine (PI) stain, acrosome integrity by fluorescein isothiocyanate and peanut agglutinine (FITC-PNA) and PI stain, plasma membrane functionality by hypo-osmotic swelling (HOS) test, and DNA denaturation by acridine orange (AO) test. There were no significant differences in the percentages of total motility, acrosome integrity, and DNA fragmentation among the extenders after thawing. Kinematics parameters showed significantly higher values in BotuCrio than in lactose extenders (P < .05). BotuCrio and lactose colostrum extender yielded significantly better rates for HOS-test, linearity, straightness, and wobble than egg-yolk extender (P < .05). However, in relation to sperm viability, lactose egg yolk extender showed significantly better results in comparison to the others seminal experimental media (P < .05). In conclusion, the incorporation of mare colostrum into cryopreservation media protected the sperm against cold-shock; therefore, it may be a good cryoprotectant agent alternative in extenders for freezing stallion semen.

A preliminary study on the use of jenny colostrum to improve quality in extenders for freezing donkey semen.

Sperm quality in donkeys (Equus asinus) after freezing thawing is still considered lower than that from other animals, including horses. The aim of this study was to test a new freezing extender supplemented with jenny colostrum on donkey sperm. After thawing, we evaluated sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional integrity by HOS-test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. Ejaculates were collected from five fertile Donkeys. Sperm samples were pooled, diluted and cryopreserved into three experimental extender groups: BotuCrio®, lactose-extender supplemented with egg yolk (20%) and lactose-extender supplemented with jenny colostrum (20%). The results demonstrated that lactose-jenny colostrum samples displayed significantly higher values in almost all parameters evaluated (p < 0.05) compared with the other two extenders after thawing (BotuCrio® and lactose-egg yolk based extender, respectively) -Total Motility, Viability, HOS test, VCL, VSL and VAP. Acrosome status, LIN, STR and WOB despite showing lower values, none of them were statistically significant (p > 0.05). In conclusion, the extender containing jenny colostrum can be successfully used for donkey semen crypreservation and could effectively improve donkey sperm qualities after freezing -thawing.

Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C.

Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4ºC and 16ºC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4ºC and 16ºC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.

Effects of seminal plasma and different cryoprotectants on rabbit sperm preservation at 16°C.

The purpose of this research was to assess whether the presence of seminal plasma (SP) can improve sperm quality of rabbit spermatozoa stored at 16°C for 72 h and moreover evaluate the cryoprotectant effects of glycerol, N-N-Dimethylformamide (DMF), and N-methyl-2-pyrrolidone (NMP). Semen samples were pooled and divided in eight fractions. Four of them were diluted with INRA (extender A), INRA with 6% glycerol (extender B), INRA with 6% DMF (extender C), or INRA with 6% NMP (extender D), respectively. The other four fractions were centrifuged, and the supernatant was removed in order to eliminate SP. Each sample was then resuspended with extender A, B, C, or D, respectively. All samples were stored at 16°C and analysed at 4, 24, 48, and 72 h by ISAS®, vitality test, HOS test, and acrosome integrity test. After analyse of the results, SP samples showed a significantly higher percentage (P=0.020) in the HOS test (71.9 ± 1.6%) than non-SP samples (66.5 ± 1.6%). Non-SP samples had better results for kinematic parameters. Extenders A and C showed great results for the percentage of motile spermatozoa (63.1 ± 4.3% and 63.4 ± 3.7%, respectively), vitality (88.9 ± 2.6% and 87.7 ± 2.7%, respectively), and HOS test (68.9 ± 1.4% and 75.2 ± 1.4%, respectively). Extenders B and D showed worse data for sperm quality. These results suggest that SP has a protective effect on rabbit sperm membranes and maintains better sperm motility. The addition of glycerol and NMP to INRA does not improve rabbit sperm quality; nevertheless, the DMF cryoprotectant exerts a protective effect on the membrane of spermatozoa, improving seminal quality during rabbit sperm preservation at 16°C.

Long-term preservation of freeze-dried rabbit sperm by adding rosmarinic acid and different chelating agents.

Freeze-drying (FD) technique has been applied as an alternative technology to preserve gene resources to allow simple sperm preservation and shipment at 4 °C. Nevertheless, DNA sperm might be damaged by mechanical or oxidative stress throughout FD procedure. Therefore, suitable protection to maintain DNA integrity is required. The aim of this study was to determine the effect of rosmarinic acid (RA) as an antioxidant and two chelating agents (EGTA and EDTA) on the DNA integrity of freeze-dried rabbit sperm after storage of the samples at 4 °C and room temperature for 8 months. Rabbit sperm were freeze-dried in basic medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with 50 mM EGTA (1), 50 mM EGTA plus 105 µM RA (2), 50 mM EDTA (3) or 50 mM EDTA plus 105 µM RA (4). Semen samples were kept at 4 °C and room temperature during 8 months. After rehydration, DNA integrity was evaluated with Sperm Chromatin Dispersion test observing that DNA fragmentation was higher when semen samples were freeze-dried with EGTA (10.9%) than with EDTA (4.1%) (p < 0.01). Furthermore, RA acted better under adverse conditions and no significant differences were found in temperature storage. Summarizing, FD is a method that can allow simple gene resources preservation among 4 °C to 25 °C during 8 months and transportation without the need for liquid nitrogen or dry ice. EDTA chelating agent is the most suitable media for freeze-dried rabbit sperm and the addition of RA protects the DNA against the oxidative stress caused during FD procedure.

Chelating agents in combination with rosmarinic acid for boar sperm freeze-drying.

The presence of DNA protective agents in the medium is necessary to maintain sperm functionality after freeze-drying procedure. The objective of this study was to investigate the effect of chelating agents, ethylene diaminetetraacetic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA), in combination with rosmarinic acid (RA) on DNA integrity of freeze-dried boar sperm. We also examined the effect of these agents on the in vitro developmental ability of porcine oocytes following sperm injection (ICSI). Heterospermic mix, obtained from ejaculated sperm of three boars, was freeze-dried in two different chelating agents' media: 50mM EDTA or 50mM EGTA, and in these media supplemented with 105µM of rosmarinic acid. Frozen-thawed sperm was used as control. After rehydration, samples were subjected to DNA damage detection using Sperm Chromatin Dispersion test. ICSI was performed to verify the ability of freeze-dried sperm to participate in embryonic development. Five replicated trials were carried out for each group. In the presence of rosmarinic acid, the percentage of spermatozoa with DNA damage decreased significantly (p=0.010), without differences between the two chelating agents combination. EDTA solution preserves more efficiently DNA integrity of boar sperm than EGTA solution (p=0.002). There were no significant differences among the studied groups related to the blastocyst formation rate. Results suggested that the addition of rosmarinic acid to the medium improves sperm DNA integrity after freeze-drying, but does not promote fertilization and blastocyst development. We also observed a similar percentage of embryos production with freeze-dried and with frozen-thawed sperm.

In vitro developmental ability of ovine oocytes following intracytoplasmic injection with freeze-dried spermatozoa.

Freeze-drying (FD) is a new and alternative method to preserve spermatozoa in refrigeration or at room temperature. Suitable protection is required to maintain the sperm DNA integrity during the whole process and storage. The aim of this study was to examine the effect of rosmarinic acid and storage temperature on the DNA integrity of freeze-dried ram sperm. In addition, we evaluated the in vitro developmental ability to the blastocyst stage of oocytes injected with freeze-dried sperm. Ram sperm was freeze-dried in basic medium and in this medium supplemented with 105 µM rosmarinic acid. The vials were stored for 1 year at 4 °C and at room temperature. Frozen sperm was used as control. After rehydration, sperm DNA damage was evaluated, observing that the percentage of spermatozoa with DNA damage decreased significantly in the presence of rosmarinic acid, without differences between the two storage temperatures. Moreover, no differences were observed between the freeze-dried group and the frozen-thawed group in terms of blastocyst formation rate. We proved for the first time that ovine spermatozoa can be lyophilized effectively, stored at room temperature for long term, reconstituted and further injected into oocytes with initial embryo development.

Fertilisation rate obtained with frozen-thawed boar semen supplemented with rosmarinic acid using a single insemination timed according to vulvar skin temperature changes.

Artificial insemination (AI) of sows with frozen-thawed semen usually results in lower pregnancy rates and litter sizes than the use of liquid preserved semen. The present study evaluated the effectiveness of vulvar skin temperature changes as a predictor of ovulation in sows and determined the fertility rates obtained after AI with frozen-thawed semen supplemented with rosmarinic acid (RA). Semen was collected from mature boars and cryopreserved in experimental extenders supplemented with or without 105 µM of RA. Multiparous sows were inseminated with a single dose of semen when vulvar skin temperature decreased to a value below 35 °C. Intrauterine insemination was performed using 1.5 × 109 spermatozoa. The sows were slaughtered 48 h after AI and the embryos and oocytes were recovered from the oviducts. Total and progressive motility, viability and acrosome integrity were significantly (P < 0.05) higher in RA-supplemented semen samples compared with the control. Fertilisation occurred in all sows inseminated in the study, although there were no significant differences between the experimental groups. Sows inseminated with RA-supplemented semen showed a slight increase in the number of embryos recovered as compared to sows inseminated with control semen. In conclusion, insemination according to vulvar skin temperature changes resulted in successful fertilisation in all sows, although supplementation of the freezing media with RA did not improve the fertilising ability of frozen-thawed boar sperm.

Rosmarinic acid improves function and in vitro fertilising ability of boar sperm after cryopreservation.

During cryopreservation, oxidative stress exerts physical and chemical changes on sperm functionality. In the present study we investigated the antioxidant effect of rosmarinic acid (RA) on quality and fertilising ability of frozen-thawed boar spermatozoa. Ejaculates collected from mature boar were cryopreserved in lactose-egg yolk buffer supplemented with different concentrations of RA (0 µM, 26.25 µM, 52.5 µM and 105 µM). Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels, DNA oxidative damage (8-hydroxy-2-deoxyguanosine base lesion) and in vitro fertilisation ability were evaluated. Total and progressive motility were significantly higher in experimental extenders with RA than in the control (P<0.05) at 0 and 120 min post-thawing. The plasma and acrosomal membrane integrity were improved by supplementation with 105 µMRA (P<0.05). Negative correlation between RA and malondialdehyde (MDA) concentration were determined (P<0.05). After thawing, the percentage of spermatozoa with oxidised DNA did not differ between extenders, however, at 120 and 240 min post-thawing, the samples supplemented with 105 µMRA showed the lowest DNA oxidation rate (P<0.05). The penetration rate was significantly higher on spermatozoa cryopreserved with 105 µMRA (P<0.05). The results suggest that RA provides a protection for boar spermatozoa against oxidative stress during cryopreservation by their antioxidant properties.

Progesterone improves porcine in vitro fertilisation system.

In an effort to improve the quality of in vitro produced porcine embryos, the effect of progestagens - progesterone analogues - on the in vitro developmental competence of porcine oocytes was studied. A total of 1421 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa. Progestagens were added to late maturation and embryo cultures (10 IU/ml). Fertilisation success (pre-maturation, penetration, monospermy and efficiency) and nuclear maturation were evaluated. There were no differences among prematuration rates between groups (P = 0.221). Penetration rates were higher (P < 0.001) in the presence of progestagens (75.0%) as compared to the control (51.7%). However, no differences were observed in monospermy percentages (P = 0.246). The results indicated that supplementation with progestagens increased the efficiency of the in vitro fertilisation system (P < 0.001). An additional beneficial effect was observed in nuclear maturation with progestagens (P = 0.035). In summary, progestagen supplementation is an important factor to improve the in vitro fertilisation procedure.

Boar sperm tyrosine phosphorylation patterns in the presence of oviductal epithelial cells: in vitro, ex vivo, and in vivo models.

Spermatozoa transport through the oviduct is a controlled process that regulates sperm capacitation. A crucial event involved in capacitation is protein tyrosine phosphorylation (TP). This study was undertaken to determine whether similarities exist in protein TP distribution between spermatozoa bound or unbound to oviductal epithelial cells (OEC) in three different conditions: i) in vitro, spermatozoa coincubated with OEC cultures; ii) ex vivo, spermatozoa deposited in porcine oviductal explants from slaughtered animals; iii) in vivo, in which sows were inseminated and the oviduct was recovered. The localization of phosphotyrosine protein was determined using indirect immunofluorescence. The distribution of protein TP was significantly (P<0.05) different between bound and unbound cell populations in all experiments. In sows inseminated close to ovulation, spermatozoa were found mainly in the utero-tubal junction, where spermatozoa exhibited higher proportion of flagellum phosphorylation. Spermatozoa not bound to OEC exhibited high levels of protein phosphorylation (phosphorylated equatorial subsegment and acrosome and/or phosphorylated flagellum) in the ex vivo and in vivo experiments (P<0.05). However, unbound spermatozoa coincubated with OEC in in vitro conditions tended to show intermediate levels of TP (equatorial subsegment with or without phosphorylated flagellum). In spermatozoa bound to OEC, protein TP was located in the equatorial subsegment or presented no phosphorylation (P<0.05). Although sperm capacitation conditions in vivo were not reproducible in vitro in our experimental conditions, sperm and OEC binding seemed to be a mechanism for selecting spermatozoa with a low level of TP in in vivo, ex vivo, and in vitro experiments.

Effect of the in vitro maturation medium on equine oocytes: comparison of follicular fluid and oestrous mare serum.

The present study evaluated the effect of supplementing the medium used to mature equine oocytes in vitro with oestrous mare serum (EMS) or horse follicular fluid (HFF). To this end, 144 ovaries were obtained from mares aged 16-21 months and transported to the laboratory in Dulbecco's phosphate buffered saline (D-PBS) at 30 degrees C. Oocytes were harvested from the ovaries by slicing, and then selected for in vitro maturation (IVM) according to the number of cumulus cell layers and the characteristics of the cytoplasm. The selected oocytes were washed three times in TCM199 medium plus HEPES (TCM-199H) or in the same medium plus glutamine (TCM-199G), then matured in vitro in six study groups established according to the in vitro maturation (IVM) treatment to see possible interactions between HEPES and glutamine on other supplements: Ten percent EMS was added to two of these media (TCM-199H+EMS and TCM-199G+EMS) and 10% HFF was added to the media in two other groups (TCM-199H+HFF and TCM-199G+HFF). IVM was performed at 38.5 degrees C for 40 h in a controlled atmosphere (5% CO2, 95% relative humidity). The findings indicate that the presence of EMS or HFF in the TCM-199H medium gives rise to the best results in terms of the proportions of oocytes reaching maturity (37.7% and 36.8%, respectively). The values obtained with EMS and HFF were statistically similar to each other but differed from the other treatments. The media containing glutamine led to the highest proportions of degenerated oocytes.