L. G. P. Bezerra

Environmental Factors Related to a Semiarid Climate Influence the Freezability of Sperm from Collared Peccaries (Pecari tajacu Linnaeus, 1758)

The influence of environmental factors in a semiarid climate on characteristics of fresh and frozen/thawed sperm collected from collared peccaries (Pecari tajacu) was assessed. Semen from 11 male collared peccaries was collected by electroejaculation during the peaks of the dry and rainy periods while rainfall indices, air temperatures, relative humidity levels, and wind speeds were measured. The number, motility, morphology, osmotic response, and membrane integrity of sperm in the collected ejaculates were assessed. Samples were then frozen in liquid nitrogen, thawed, and reassessed. The rainfall index of the rainy period (73.2 mm) was significantly higher than that of the dry period (13.6 mm) and the relative humidity was significantly higher during the rainy period (74.6%) than it was during the dry period (66.8%). Air temperature and wind speed did not differ between the two periods. Characteristics of sperm in the fresh samples were not affected by environmental parameters. In contrast, computerized analysis revealed that sperm in samples frozen during the rainy period exhibited better post-thaw membrane integrity (28.6 ± 6%), motility (29.5 ± 7.7%), and rapid sperm population (13.7 ± 6.2%) than did sperm in samples frozen during the dry period (23.4 ± 3% membrane integrity, 14.6 ± 4.1% motility, and 4.1 ± 1.2% rapid sperm; p < 0.05). Other characteristics of the frozen/thawed sperm did not differ depending on the period in which they were collected. We demonstrated that environmental parameters did not affect the quality of fresh sperm, but could influence the freezability of sperm collected from collared peccaries raised under a semiarid climate.

Epididymal sperm from Spix’s yellow-toothed cavies sperm successfully cryopreserved in Tris extender with 6% glycerol and 20% egg yolk

As a non-threatened hystricognath rodent species, Spixs yellow-toothed cavies can be used as a model for the development of assisted reproductive techniques for the conservation of closely related species. The objective was to establish a functional protocol for cryopreservation of epididymal sperm from these cavies. Twelve sexually mature males, ∼2 y old and weighing ∼300 g, were euthanized. Sperm were recovered by retrograde flushing of the vas deferens and cauda epididymis with Tris extender. Thereafter, sperm were extended in Tris plus 20% egg yolk, with 3%, 6% or 9% glycerol or dimethyl sulfoxide (DMSO), placed in 0.25 mL straws and cryopreserved in liquid nitrogen. Sperm concentration, motility (using computer-assisted sperm analysis; CASA), plasma membrane integrity, osmotic response, morphology and sperm binding-ability were determined in fresh and frozen-thawed sperm. For most sperm endpoints, glycerol was a more desirable cryoprotectant than DMSO. Data (mean ± SEM) were similar with use of 3%, 6%, and 9% glycerol (P > 0.05) in osmotic response (40.66 ± 6.3%, 42.5 ± 7.1%, and 39.5 ± 5.0% respectably), and membrane integrity (55.17 ± 5.5%, 68.4 ± 4.1%, and 59.1 ± 4.9% respectably). Among concentrations assessed, the use of 6% glycerol resulted in the greatest (P < 0.05) post-thaw values for total motility (60.9 ± 4.4%), rapid subpopulation motility (27.7 ± 3.1%) and sperm-binding capability (227.0 ± 20.2). In conclusion, epididymal sperm from the Spixs yellow-toothed cavies (G. spixii) are optimally cryopreserved in Tris extender with 6% glycerol and 20% egg yolk.

Development of fresh and vitrified agouti ovarian tissue after xenografting to ovariectomised severe combined immunodeficiency (SCID) mice

The aim of the present study was to evaluate the development of fresh and vitrified agouti ovarian tissue after xenografting to C57Bl/6 severe combined immunodeficiency (SCID) female mice. Ovaries were obtained from five female agoutis and divided into 16 fragments. Five fragments were transplanted immediately to ovariectomised SCID mice and the others were vitrified, stored for 2 weeks and transplanted only after rewarming. Tissue fragments were transplanted under the kidney capsule in recipients. The return of ovarian activity in recipients was monitored by the observation of external signs of oestrus and vaginal cytology over a period of 40 days after transplantation, after which the grafts were removed and evaluated for morphology, cell proliferation and the occurrence of DNA fragmentation. Ovarian activity returned in four of five mice that received fresh ovarian tissue from agoutis and in one of six mice that had received vitrified tissue a mean (±s.e.m.) 20.6±8.6 days after xenotransplantation. After graft removal, a predominance of primordial and primary follicles was observed in all grafts. Vitrification reduced cell proliferation and increased the occurrence of DNA fragmentation in grafted agouti ovarian tissue. In conclusion, the present study demonstrates that xenografted agouti ovarian tissue, fresh or vitrified, is able to promote the return of ovarian activity in ovariectomised SCID C57B1/6 mice. However, improvements to vitrification protocols for agouti ovarian tissue are necessary.

Characterization of the ovarian preantral follicle populations and its correlation with age and nutritional status in Brazilian Northeastern donkeys (Equus assinus)

This study aimed to characterize the ovarian follicular population and to determine its correlations with the age and nutritional status of donkeys of the Northeastern Brazilian breed. A total of 10 females with a mean age of 5.1±3.2years were submitted to ovariectomy by videolaparoscopy to obtain the ovaries. In the laboratory, the ovaries were sectioned into 6-12 fragments of approximately 7mm in diameter, which were fixed in Carnoy, dehydrated in increasing concentrations of alcohol (85%, 95% and absolute), clarified using xylol and embedded in blocks of histological paraffin. The blocks were cut in sections of 7μm and each 120th section was mounted on a slide for observation using optical microscopy. The follicle counting and identification allowed the characterization of the population of the preantral follicles. A total of 21.135±10.821 preantral follicles was counted, of which, 91.3% were primordial, 8.3% were primary follicles and 0.4% were secondary follicles. There were no differences between the two ovaries of each animal regarding the follicular population (P>0.05). There was a rate of 9.77% degenerated follicles. Values of 0.99% follicles containing two oocytes were also identified and classified as multi-oocyte follicles, always of the primordial category. The thickness of the granulosa cell layer was 1.85μm±1.39, 3.56μm±2.08 and 21.85μm±17.27, for primordial, primary and secondary follicles, respectively. There was a significant inverse correlation (r=-0.66; P<0.001) between the age of the animals and the population of ovarian follicles. A negative influence of the weight and body score on the ovarian follicular population was also observed, when donkeys had very little or a great amount of body condition. This is the first study to describe the morphometric characteristics and estimation of the population of preantral ovarian follicles in Northeastern Brazilian donkey, showing that number of preantral follicles decreased with increasing age of the animals and this finding may be affected by nutritional status of the animals.