Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.     

Behavioral and physiologic responses to environmental enrichment in the maned wolf (Chrysocyon brachyurus)

The ex situ population of maned wolves is not self‐sustaining due to poor reproduction, caused primarily by parental incompetence. Studies have shown that environmental enrichment can promote natural parental behaviors in zoo animals. The objective of this study was to determine the effects of environmental enrichment on behavioral and physiological responses of maned wolves. During an 8‐week experimental period, daily behavior observations and fecal sample collection were conducted on four adult wolves (2.2) individually housed in environments without enrichment. After 2 weeks, the wolves were chronologically provided with 2‐week intervals of hiding dead mice around the exhibit, no enrichment, and introduction of boomer balls. Responses of the wolves to enrichment were assessed based on activity levels and exploratory rates, as well as the level of corticoid metabolites in fecal samples collected daily throughout the study period. Providing wolves with environmental enrichment significantly increased exploratory behaviors (P<0.05), especially when mice were hidden in the enclosure. Fecal corticoid concentrations were increased during periods of enrichment in males (P<0.05), but not in females. Overall, there were no correlations between behavioral responses to enrichment and fecal corticoid levels. Behavioral results suggest that environmental enrichment elicits positive effects on the behavior of captive maned wolves. There is evidence suggesting that providing animals with ability to forage for food is a more effective enrichment strategy than introducing objects. There is need for a longer term study to determine the impact of environmental enrichment in this species. Zoo Biol 26:331–343, 2007. © 2007 Wiley‐Liss, Inc.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

Differences among dogs in response of their spermatozoa to cryopreservation using various cooling and warming rates

Spermatozoa collected from the caudae epididymides of 16 dogs of various breeds were suspended in an isotonic salt solution (DIMI medium) containing 0.6 M glycerol, frozen in liquid nitrogen, and their "survival" was measured after thawing. In the first experimental series, duplicate samples of spermatozoa from each of 11 dogs were cooled at rates of 0.5, 3, 11, 58, or 209 degrees C/min, stored in liquid nitrogen, and the frozen samples warmed at approximately 830 or at 33 degrees C/min. Sperm "survival" was judged by microscopic assessments of motility and of membrane integrity, the latter as assayed with Fertilight, a double fluorescent stain. Motility of frozen spermatozoa that were thawed rapidly, averaged for 11 dogs, was low at low rates, increased to a maximum at 11 degrees C/min, and then decreased significantly at higher rates (P<0.01). This inverted V-shaped curve was also observed with slow thawing, although the apparent optimum cooling rate ranged from 3 to 11 degrees C/min. The integrity of sperm plasma membranes showed a similar dependence on cooling rate, although the percentages of spermatozoa with intact plasma membranes were higher than the percentages of motile spermatozoa. Motility of spermatozoa, as a function of cooling rate, varied considerably from male to male (P<0.01), whereas membrane integrity was much more consistent among the 11 dogs. In the second experimental series with spermatozoa from 5 dogs, motility of spermatozoa frozen at 0.5 degrees C/min and warmed at 3.6, 33, 140, or 830 degrees C/min also exhibited an inverted V-shaped survival curve, in this case as a function of warming rate. In summary, high survival of frozen-thawed canine epididymal spermatozoa depended on both cooling and warming rates, but spermatozoa from each dog exhibited their own sensitivity to cooling and warming rates.     

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.     

Abnormal reproductive patterns in Przewalski's mares are associated with a loss in gene diversity

The ex situ population of the Przewalski's horse (Equus ferus przewalskii) is not self-sustaining (20% foaling rate), and the demography is skewed toward aging individuals with low gene diversity. We designed the present study to gain a better understanding of the reproductive biology of the Przewalski's mare and to determine whether age and gene diversity influenced reproductive function. Urine samples were collected 3-7 days/wk from 19 mares from May to September, and ultrasound examinations of follicular structures were performed 3 days/wk for 5 wk from May through July in nine individuals. A high proportion of mares exhibited abnormal (endocrine, 5 [26.3%] of 19; follicular, 2 [22.2%] of 9) or acyclic (endocrine, 4 [21.1%] of 19; follicular, 3 [33.3%] of 9) reproductive patterns. In four cyclic mares, estrous cycle length was 25.1 ± 1.2 days, with 12.2 ± 0.9 days of diestrus. Follicles in cyclic mares grew 1.2 ± 0.6 mm per day and ovulated after reaching 40.4 ± 8.9 mm. Mares with a high coefficient of inbreeding excreted reduced levels of mean urinary estrogens (r(2) = 0.476, P < 0.05), but age had no significant impact on reproductive patterns in this population. Overall, these data suggest that long-term genetic management of this population is necessary to maintain reproductive fitness.     

Effect of ambient temperatures during oocyte recovery on in vitro production of bovine embryos

Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.     

Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury,but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me₂SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me₂SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to <600 mOsm NaCl or sucrose was less (P < 0.05) than fresh ejaculate, but comparable (P > 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1 M glycerol or Me₂SO and cooled from 5 °C to −120 °C at −57.8 °C, −124.2 °C or −67.0 °C/min, frozen in LN₂, thawed in a water bath for 30 s at 37 °C or 10 s at 50 °C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P < 0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P < 0.05) post-thaw motility (20.0 ± 1.9% versus 13.5 ± 2.1%) and membrane integrity (51.2 ± 1.7% versus 41.5 ± 2.2%) were observed in samples cryopreserved in Me₂SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8 °C/min being advantageous (P < 0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me₂SO as a cryoprotectant.     

Effect of treatment with recombinant bovine somatotropin on responses to superovulatory treatment in swamp buffalo (Bubalus bubalis)

The objective of this study was to determine the effect of treatment with recombinant bovine somatotropin (rBST) on the response to superovulatory treatment in swamp buffalo. Estrous cycles of 16 buffalo cows were synchronized by intravaginal administration of progesterone and estradiol benzoate, and the cows were then randomly divided into 2 groups. The rBST-treated group received 250 mg of a sustained-release formula of rBST on Day 4 after progesterone implantation, whereas the control group did not receive rBST. Both groups were then given a superovulatory regimen of twice daily injections of FSH for 3.5 d (total dose of 260 mg, i.m.), between Days 9 and 11 after administration of progesterone. The cows were bred naturally 1 d after the last FSH injection, then 6 d after breeding they were slaughtered, and their reproductive tracts were removed. The numbers of corpora lutea (CL) and follicles were recorded, and embryos were flushed out of the uterine horns. There were no significant differences between the rBST-treated and control cows for the mean numbers (+/- SEM) of CL (6.0 +/- 2.2 vs 4.3 +/- 1.1), follicles (15.9 +/- 4.1 vs 19.8 +/- 2.9), or total embryos recovered per collection (4.5 +/- 1.6 vs 2.3 +/- 1.0). However, there were significant differences between rBST-treated and control cows for the numbers of transferable embryos per collection (3.0 +/- 1.0 vs 0.8 +/- 0.3; P < or = 0.05) and the overall proportion of transferable embryos (75 vs 33%; P < or = 0.01). The results of this study show that pretreatment of swamp buffalo with rBST significantly increases the production of transferable embryos in response to superovulation.