Renato Campanarut Barnabe

Evaluation of synchronization of ovulation for fixed timed insemination in buffalo (Bubalus bubalis)

The objective of this study was to evaluate the efficiency of synchronization ovulation protocol (GnRH/PGF2a/GnRH) for fixed timed insemination in buffalo. In Experiment 1, thirty-three female buffaloes with 60 days postpartum, were divided in two groups for evaluation of the follicular dynamics (Group 1, n = 16, 10mg of GnRH/15mg of PGF2a/10mg of GnRH; Group 2, n = 17, 20mg of GnRH/15mg of PGF2a/10mg of GnRH). Ultrasonography exams were performed every 12 hours to verify the ovarian response to the first GnRH injection. Another evaluations were performed on PGF2a injection and second GnRH injection. After last hormonal injection, ultrasonography exams were performed every 6 hours to verify the moment of the ovulation. Blood samples were collected on Days 0, 7 and 9 to evaluate the progesterone concentrations at the moment of the hormonal injections. In Experiment 2, the efficiency of the synchronization of ovulation was verified for fixed timed insemination (16 hours after second GnRH injection, n=1053), during the favorable and unfavorable reproductive season, in the years of 1998 and 1999. Animals received 20mg of GnRH/15mg of PGF2a/10mg of GnRH. Besides, also recorded and evaluated aspects like body condition score, parity, postpartum period, year and the reproductive season (favorable or unfavorable season), on the conception rate. In Experiment 1, it was verified that 33.0 ± 8.3h after the first GnRH injection, 60.60% of the animals ovulated (Group 1 = 70.5 vs. Group 2 = 50.00%; P>0.05). The dose of GnRH (10mg vs. 20mg) and the progesterone concentration did not interfere in ovulation in the beginning of the treatment (P>0.05). Animals that ovulated (n = 20) after the first GnRH injection presented larger follicular diameter (P 0.05). In Experiment 2, it was observed conception rate of 45.40% (n = 1053). Animals synchronized during the favorable reproductive season presented better conception rate than the unfavorable season (48.80% vs. 6.90%; P 0.05). In summary, the Experiments indicated that the use of synchronization of the ovulation for fixed timed insemination, during the favorable reproductive season, presents satisfactory results in buffalo and could still be better when used multiparous buffaloes with good body conditon score in the beginning of the treatment.

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20 degrees C until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37 degrees C for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 +/- 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350% above baseline). On one occasion, we did not detect any variation. Mean (+/- SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 +/- 26.7, after: 213.7 +/- 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

Puberty and growth rate in thoroughbred fillies

We measured progesterone and estradiol levels from birth to the beginning of adult life in 10 Thoroughbred fillies from the Equilia Stud Farm in Avaré SP, Brasil. The animals were measured and weighed monthly for the determination of body development and of a possible correlation between the rate of weight and height gain and the onset of detectable sex hormone levels. Jugular blood was collected twice a week and stored at -20 degrees C until assay of progesterone by a solid phase RIA with a sensitivity of 0.32 nmol L and of estradiol by liquid phase RIA adapted to low levels (3.67 pmol L ). The fillies were born with high serum levels of both hormones, which fell to undetectable levels by the first week of life. A variation in growth rate was observed, with the highest levels occurring from birth to the 3rd month of life, followed by a reduction until 15 mo of life, when fast growth was resumed. The monthly weight gain was 1.5% when the fillies reached puberty and 5.4% during the next month, (P < 0.05, Friedman test). During this second period of accelerated growth after the beginning of progesterone production at detectable levels (above 0.318 nmol L ), the parameters of skeletal growth did not differ (P > 0.05). The month of onset of puberty was the month of lowest weight gain in the life of the fillies, and it coincided with the highest insolation period. In conclusion, horses, like all other developed vertebrates, have a double pattern of development, with the acceleration observed at puberty depending on sex steroids, which in turn coincides with the highest insolation period. Gonadal activity characterized by serum progesterone levels was low from birth to the onset of puberty. After puberty the progesterone cycles were similar to those of adult animals with a mature hypothalamic-gonadal axis.

Biometria testicular de touros Nelore (Bos taurus indicus) e touros cruzados Nelore-europeu (Bos taurus indicus x Bos taurus taurus) aos 20 e 24 meses de idade

The objective of the present study was to evaluate the effects of age and genetic group in bulls, and determine the relationship between these two factors on testicular biometry (scrotal circunference and testicular consistency). One hundred and ninety Nellore Bulls and 447 crossed Nellore-European bulls were used when aged 20 and 24 months. Scrotal circunference and testicular consistency were found to be influenced by genetic group, age and genetic group and age interaction. Higher scrotal circunference and testicular consistency were fond in crossed bulls as compared to Nellore ones, both at 20 and 24 months of age.

Analysis of some normal parameters of the spermiogram of captive capuchin monkeys (Cebus apella Linnaeus, 1758 )

Nine adult males of capuchin monkey (Cebus apella) were electro-ejaculated with a rectal bipolar probe under general anaesthesia with tiletamine -zolazepan association. From the obtained ejaculate, we only analysed the liquid fraction in order to avoid any kind of chemical treatment to attain dissolution of the seminal coagulum. The mean obtained volume was 0,2ml with the sperm concentration of 56.169 x 106 sperm/ml and mean motility 68,4% and vigor 2,6. The mean percentage of morphologic normal spermatozoa was 39%. The technique was efficient for semen collection in Cebus apella and allowed seminal evaluation without possible sperm lesions caused by chemical treatment for coagulum dissolution.

Avaliaçäo da capacidade de penetraçäo de sêmen congelado de onça pintada (Panthera onca) em oócitos heterólogos

Assisted reproductive technologies can be viewed as one potential approach for safeguarding wild species. In this study were evaluated the fertility of captivity male jaguar (Panthera onca) and different capacitation media using the golden hamster zona free oocyte penetration assay. We used frozen/thawed semen from 3 animals housed at Bosque dos Jequitibas, Campinas/SP, to test the Percoll gradient, Swim-up and Swim-up + 1 h incubation (5% CO2 / 38°C), considering as penetration the spermatozoa head descondensation visualized within the oocyte. The results rate was greater for Percoll (26.5%) as compared to Swim-up (8.1%) (X2 = 19.93; p < 0.05). It was not observed penetration with Swim-up + 1 h incubation (5% CO2 / 38°C). It is concluded that Percoll and Swim-up are efficient methods to perform the golden hamster zona free oocyte penetration assay using frozen/thawed jaguar (Panthera onca) spermatozoa. The low rate of penetration could be related to the high rate of morphological abnormal spermatozoa observed in the samples examined.

Citologia aspirativa por agulha fina (CAAF), em testículo de onça pintada (Panthera onca), utilizada como ferramenta no diagnóstico de infertilidade

Several methods of testicular punch biopsy were proposed for obtaining material for histologic or cytologic evaluation, but did not received enough clinical acceptance because it was considered to be too traumatizing. The fine Needle Aspiration (FNA) is considered a useful, simple and fast method to obtain samples from tissues. Regarding the importance of FNA in wild animals, this technique was tried in captive adult jaguar (Panthera onca) aiming the evaluation and possible causes of infertility. Using a needle and disposable syringe, the testis were aspirated. The whole aspirated was smeared onto a microscope slide and stained with Diff-quick method. Semen samples were collected by electroejaculation and analyzed for pH, total volume, motility, status, total spermatozoa count and morphology. Evaluation of sperm volume, pH, motility vigor and morphology were normal. However, was found below concentration. Cytologic quantification revealed germinative cells in all testicles. Among serial types of morphological normal spermatogenesis cells, there were found theratological forms of double final spermatids. These anomalous forms of final spermatids have not been noticed yet in FNA papers. So, we concluded that FNA together with other techniques, provides a useful tool in male infertility diagnostics, mainly when related to endangered species.

Semen evaluation of Murrah buffalo bulls using sperm functional tests

Abstract The aim of this experiment was to evaluate membrane integrity, vitality, and mitochondrial cytochemical activity, in frozen semen samples of buffalo bulls and compare those functions with the routine semen evaluation and field fertility. Twenty one frozen semen batches from 2 buffalo bulls were used for AI. For the semen evaluation, after thawing, an aliquot was evaluated for motility and vigor. An aliquot of each batch was used to evaluate the cytochemical activity using the 3-3’ diamino benzidine. Samples were scored in four classes according to the degree of midpiece staining, being class I showing midpiece totally stained, indicating full mitochondrial activity, and class IV showing no staining of the midpiece, indicating no mitochondrial activity. Two other aliquots were used for the hypo-osmotic swelling test (HOST) and the eosin nigrosin staining (VIT), to evaluate membrane integrity and vitality, respectively. Correlations were found between pregnancy rate and vitality and class II and III of the DAB staining (r=0.53, r=-0.39, and r=-0.38, respectively; p=0.05). No correlation was found for pregnancy rate and motility or vigor. Results indicate that functional tests may be an alternative to better predict the fertility of buffalo frozen semen samples.

Seasonal Variation in the Testicular Volume of Capuchin Monkeys (Cebus apella) in Captivity

The study of the reproductive strategy developed by different species in order to adapt to their environmental conditions and their meaning in an evolutionary perspective is essential for understanding the mechanisms involved in the process of reproduction. Non-human primates are very interesting models for this purpose. Some species show a typical seasonal reproductive pattern, such as rhesus macaques (Macaca mulatta) [Sade, 1964; Conaway and Sade, 1965] and ring-tailed lemurs (Lemur catta) [Zuckerman, 1953], while others, such as gorillas (Gorilla gorilla) and chimpanzees (Pan troglodytes) [Puschmann, 1975], show relative independence of the environment. Neotropical primates display many different breeding strategies. Female capuchin monkeys (Cebus apella), for instance, having reached puberty, have fertile cycles year-round [Hamlett, 1939; Nagle and Denari, 1983]. Interestingly, there are reports of a clear peak of births in free-living [Hamlett, 1939] and captive colonies [Welker et al., 1983] in the dry season, i.e. May-June in the southern hemisphere. Some authors suggest that a seasonal variation in spermatogenesis could explain the birth peak [Freese and Oppenheimer, 1981]. The aim of this study was to investigate this theory, measuring seasonal variation in total testicular volume in a captive group of male capuchin monkeys and assessing its temporal correlation with the birth season as an indirect indication of variation in male fertility.

Detecção de estro em jaguatirica (Leopardus pardalis) utilizando citologia vaginal

Utilizou-se a citologia vaginal por meio de diferentes metodos de coloracao para deteccao de cio em jaguatirica, Leopardus pardalis, pela estimulacao hormonal exogena e pela avaliacao das estruturas ovarianas por videolaparoscopia. Cinco femeas foram tratadas com eCG/hCG e FSH/LH a cada quatro meses pelo periodo de dois anos. Videolaparoscopia foi realizada apos cada tratamento utilizando-se cetamina-xilazina e isoflurano. Esfregacos vaginais foram obtidos 15 dias antes e apos a videolaparoscopia. As lâminas foram analisadas ao microscopio de luz quanto aos tipos celulares predominantes. Todos os animais apresentaram foliculos maduros (>2mm) e corpos luteos recentes em todas as intervencoes. Nao houve diferenca significativa entre os resultados obtidos na mesma coloracao de acordo com os tratamentos utilizados. Todas as tecnicas mostraram-se eficientes na deteccao de celulas superficiais queratinizadas anucleadas e nucleadas, intermediarias, parabasais e basais. Foi possivel determinar a fase de estro em Leopardus pardalis por meio da citologia vaginal