G. Lopes

Differences in preservation of canine chilled semen using different transport containers

In the present study, the effect of three different containers in the preservation of dog chilled semen, during 24, 48 and 72h was evaluated. Weekly sperm pools of different dogs were obtained, during 10 consecutive weeks. Semen samples were diluted in egg-yolk-Tris-fructose extender and stored in a Styrofoam box, a common Thermos flask and an Equitainer. Progressive motility, morphology and sperm membrane integrity were examined in semen aliquots taken daily from each container during the 3 days of storage. Additionally, integrity of the acrosome and sperm plasma membranes, determined by PI/Fitc-PSA staining was assessed at 48 and 72h of storage. At 24h no differences were observed between the three containers for the evaluated parameters. At 48h samples kept in the Equitainer presented a higher progressive motility than samples kept in the Thermos. At 72h, progressive motility was higher in the Equitainer than in the other two containers. Only samples kept in the Equitainer maintained similar levels of progressive motility between 24 and 72h. Membrane integrity assessed by eosin-nigrosin deteriorated over the 72h period, whereas functional membrane integrity determined by the hypoosmotic swelling test was independently affected by type of container (the Equitainer) kept a higher percentage of sperm cells with intact membrane) and time of storage (a decrease of membrane integrity between 24 to 72h). Staining with PI-Fitc-PSA allowed the detection of differences between containers but not between the two studied storage periods (48 and 72h). The results indicated that the use of the Equitainer is preferable when transporting chilled dog semen for more than 48h.

Characteristics of stallion epididymal spermatozoa at collection and effect of two refrigeration protocols on the quality of the frozen/thawed sperm cells

Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the extended epididymal sperm cells. Ejaculated sperm samples were obtained from 10 healthy stallions with at least 15 days of sexual rest, before routine orchiectomies. Spermatozoa were recovered from the epididymal tail immediately after castration (EPI), after refrigeration of the epididymis for 24h at 4°C (EPI R) and recovered from epididymal tail immediately after castration and stored for 24h at 4°C (EPI RR). Total motility, straight-line velocity, percentage of rapid cells, viability and morphological defects were similar (p>0.05) among different treatments, and post-thaw viability was higher (p<0.05) in EPI than in the ejaculated sperm. The similarity of post-thaw parameters led us to conclude that immediate collection and refrigeration of the epididymal sperm cells or refrigeration of the whole epididymis are equally efficient as a means of transporting material for 24h before cryopreservation of epididymal spermatozoa.

Tris–Egg Yolk–Glycerol (TEY) Extender Developed for Freezing Dog Semen is a Good Option to Cryopreserve Bovine Epididymal Sperm Cells

Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis-epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze-thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed(®) or a Tris-egg yolk (TEY)-based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin-nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post-thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed(®) . Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed(®) . We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post-thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.

DNA fragmentation in canine oocytes after in vitro maturation in TCM-199 medium supplemented with different proteins

In the present study, the effect of different protein supplementation on meiotic nuclear configuration, DNA fragmentation (TUNEL assay) and metabolic parameters of dog oocytes cultured in vitro for 72 h was investigated. TCM-199 medium was supplemented either with 0.3% bovine serum albumin (BSA) or with 10% bitch heat inactivated plasma (OBP) collected before the LH peak or with OBP collected between the LH peak and ovulation or OBP collected after ovulation. After culture, more than 70% of the cumulus-oocyte complexes cultured in plasma groups presented extensive cell expansion, while none of those cultured in BSA showed extensive expansion of the cumulus (P < 0.05). Glucose consumption and lactate production was lower (P < 0.05) in the BSA-supplemented medium than in plasma-supplemented groups. In all groups, high amounts of alanine were produced. A higher number of oocytes with DNA fragmentation were observed in the BSA group, while in the plasma-supplemented groups more oocytes presented undistinguishable nuclear material. Only a small percentage of the oocytes (7.4-12.7%) had intact DNA after culture and within these, no differences were observed between groups in number of oocytes at each chromatin configuration stage. No differences in the percentage of oocytes reaching metaphase II (MII) were observed between experimental groups. Still, only 2% of cultured oocytes reached MII, but 85.7% of these had intact DNA. Conversely, all other chromatin configurations presented a high proportion of fragmented DNA (germinal vesicle 79.8%; meiosis resumption 73.3%; unclassified 95.2%). In conclusion, a high percentage of canine oocytes that do not complete meiotic maturation to MII are degenerated, whereas a high proportion of MII oocytes have intact DNA, independently of the protein supplement used.

Comparative ultrastructural analysis of diestrous and anestrous canine Grade 1 cumulus-oocyte complexes

The objective of the present study was to characterize, by means of transmission electron microscopy, immature Grade 1 cumulus-oocyte complexes (COCs) obtained from ovaries collected from bitches at diestrus and anestrus, after routine ovariohysterectomy. Cumulus-oocyte complexes were recovered after slicing the ovarian cortex and Grade 1 COCs were selected and prepared for transmission electron microscopy. All oocytes were at germinal vesicle stage in two different configurations. In 29 out of 37 COCs, oocytes presented a central or eccentric located nucleus (GVc), frequently containing a reticulated nucleolus, with a predominance of profound follicular cell processes (FCP), abundant endoplasmic reticulum (ER) and a moderated number of lipid droplets. In eight out of 37 COCs, oocytes had a peripheral located nucleus (GVp) containing small compact nucleolus, a thin perivitelline space, both superficial and profound FCP, moderate ER content and abundant lipid droplets. A higher (p<0.05) proportion of GVp was found in oocytes collected in diestrus (36.8% - 7 out of 19) than in anestrus (5.6% - 1 out of 18). In addition, distinct ultrastructure characteristics among GVc associated with estrous phase were noted. Furthermore, a number of structures, some of which had not been reported before, were present in canine prophase I oocytes at variable frequencies. The evident variation in the presence, quantity and distribution of cell organelles in canine immature Grade 1 oocytes is discussed in relation to the biological phase of the oocyte and the reproductive stage of the donor bitch.