Partial luteolysis during early diestrus in cattle downregulates VEGFA expression and reduces large luteal cell and corpus luteum sizes and plasma progesterone concentration.

Our objectives were to investigate potential changes in the size of steroidogenic large luteal cells (LLC) during partial luteolysis induced by a sub-dose of cloprostenol in early diestrus and to determine transcriptional variations in genes involved in corpus luteum (CL) functions. Cows were subjected to an Ovsynch protocol, with the time of the second GnRH treatment defined as Day 0 (D0). On D6, cows were randomly allocated into three treatments: Control (2 mL saline, im; n = 10), 2XPGF (two doses of 500 µg of cloprostenol, im, 2 h apart; n = 8) or 1/6PGF (single dose of 83.3 µg of cloprostenol, im; n = 10). Before treatments and every 8 h during the 48-h experimental period, blood samples were collected and CL volumes measured. Furthermore, two CL biopsies were obtained at 24 and 40 h post-treatment. The 1/6PGF treatment caused partial luteolysis, characterized by sudden decreases in plasma progesterone (P4) concentrations, luteal volume and LLC size, followed by increases (to pretreatment values) in P4 and luteal volume at 24 and 40 h post-treatment, respectively. However, at the end of the study, P4, luteal volume and LLC size were all significantly smaller than in Control cows. Temporally associated with these phenotypes, there was a lower mRNA abundance of VEGFA at 24 and 40 h, and ABCA1 at 24 h (P < 0.05). In conclusion, a sudden reduction in CL size during partial luteolysis induced by a sub-dose of PGF2α analog on day 6 of the estrous cycle was attributed to a reduction in LLC size, although these changes did not account for the entire phenomenon. In addition to its involvement in reducing CL size, decreased VEGFA mRNA abundance impaired CL development, resulting in a smaller luteal gland and lower plasma P4 concentrations compared to Control cows.

Testicular hyperthermia reduces testosterone concentrations and alters gene expression in testes of Nelore bulls.

Increased testicular temperature reduces sperm motility, morphology and fertility. Our objectives were to characterize effects of testicular hyperthermia (scrotal insulation) on acute testosterone concentrations and gene expression in Bos indicus testes. Nelore bulls (n = 20), ∼27 mo of age, 375 kg, scrotal circumference >31 cm, with ≥30% motile sperm, were allocated into four groups (n = 5/group): non-insulated (Control) and insulation removed after 12, 24, or 48 h. Immediately after insulation, intratesticular temperatures (needle thermocouples) were coolest in Control bulls and warmest in 48-h bulls (mean ± SEM, 35.28 ± 0.31 vs 38.62 ± 0.57 °C, P < 0.05). Bulls were castrated and testes recovered. Testicular testosterone concentrations were higher in Control versus 48-h bulls (3119 ± 973.3 and 295.5 ± 122.8 ng/g of tissue, respectively, P < 0.05). Total RNA was extracted, reverse transcribed and RT-qPCR done. For STAR, mRNA abundance decreased from Control to 48 h (1.14 + 0.32 vs 0.32 + 0.5, P < 0.05). For BCL2, expression decreased from Control to 24 h (1.00 + 0.07 vs 0.70 + 0.12, P < 0.05), but then rebounded. In addition, GPX1 had a 70% increase (P < 0.05) at 48 h, whereas HSP70 had a 34-fold increase (P < 0.05) at 12 h and 2- and 14-fold increases (P < 0.05) at 24 and 48 h, respectively. HSF1, BAX, P53 and CASP 8 remained unchanged. Downregulation of STAR, critical in androgen production, was consistent with reduced testosterone concentrations, whereas increased GPX1 enhanced testicular antioxidative capability. Huge increases in HSP70 conferred protection again apoptosis and cell destruction, whereas reduced BCL2 promoted apoptosis. These findings provided novel insights into acute tissue responses (testosterone and gene activity) to testicular hyperthermia in B. indicus bulls.

Short communication: Heat stress does not affect induced luteolysis in Holstein cows.

Heat stress (HS) has deleterious effects on bovine reproduction, including prolongation of the luteal phase in Holstein cows, perhaps due to compromised luteolysis. The objective was to characterize effects of HS on luteolytic responses of nonlactating Holstein cows given 25 or 12.5 mg of PGF2α on d 7 of the estrous cycle. Cows were randomly distributed into 2 environments: thermoneutral (n = 12; 25°C) or HS (n = 12; 36°C). In each environment, cows were treated with 2 mL of saline, 25 or 12.5 mg of PGF2α (n = 4 cows per group). The HS environment induced a significant increase in rectal temperature and respiratory rate compared with the thermoneutral environment. Heat stress did not have significant effects on luteolytic responses or circulating progesterone concentrations. Rapid and complete luteolysis occurred in all cows given 25 mg of PGF2α and in 4 of 8 cows given 12.5 mg; the other 4 cows given 12.5 mg had partial luteolysis, with circulating progesterone concentrations initially suppressed, but subsequently rebounding. Therefore, we conclude that HS does not change corpus luteum sensitivity to PGF2α.

Short-term testicular warming under anesthesia causes similar increases in testicular blood flow in Bos taurus versus Bos indicus bulls, but no apparent hypoxia.

Bull testes must be 4-5 °C below body temperature, with testicular warming more likely to cause poor-quality sperm in Bos taurus (European/British) versus Bos indicus (Indian/zebu) bulls. Despite a long-standing dogma that testicular hyperthermia causes hypoxia, we reported that increasing testicular temperature in bulls and rams enhanced testicular blood flow and O2 delivery/uptake, without hypoxia. Our objective was to determine effects of short-term testicular hyperthermia on testicular blood flow, O2 delivery and uptake and evidence of testicular hypoxia in pubertal Angus (B. taurus) and Nelore (B. indicus) bulls (nine per breed) under isoflurane anesthesia. As testes were warmed from 34 to 40 °C, there were increases (P < 0.0001, but no breed effects) in testicular blood flow (mean ± SEM, 9.59 ± 0.10 vs 17.67 ± 0.29 mL/min/100 g, respectively), O2 delivery (1.79 ± 0.06 vs 3.44 ± 0.11 mL O2/min/100 g) and O2 consumption (0.69 ± 0.07 vs 1.25 ± 0.54 mL O2/min/100 g), but no indications of testicular hypoxia. Hypotheses that: 1) both breeds increase testicular blood flow in response to testicular warming; and 2) neither breed has testicular hypoxia, were supported; however, the hypothesis that the relative increase in blood flow is greater in Angus versus Nelore was not supported. Although these were short-term increases in testicular temperature in anesthetized bulls, results did not support the long-standing dogma that increased testicular temperature does not increase testicular blood flow and an ensuing hypoxia is responsible for decreases in motile, morphologically normal and fertile sperm.

Expression of fibroblast growth factor 22 (FGF22) and its receptor, FGFR1B, during development and regression of bovine corpus luteum.

The aim of this study was to determine the expression of fibroblast growth factor 22 (FGF22) in the bovine corpus luteum (CL) and to investigate the effects of in vivo total or partial cloprostenol-induced luteolysis on the mRNA abundance of FGF22 and its receptor, FGFR1B. Corpora lutea at different stages of development were then dissected from abattoir ovaries (n = 10/stage); a portion of the tissue samples was fixed in paraformaldehyde and the remaining samples were homogenized and subjected to total RNA extraction. To assess mRNA abundance of target genes during induced luteolysis, nineteen cows were synchronized and then randomly assigned to a Latin square design as follows: Control; 2 administrations of prostaglandin F2α (PGF2α, total luteolysis; 2 × 250 µg of cloprostenol sodium) and 1/6PGF2α (partial luteolysis; 83.33 µg of cloprostenol sodium). FGF22 and FGFR1B expression levels were measured by RT-qPCR, and FGF22 protein expression was detected by immunohistochemistry. In summary, FGF22 mRNA was detected at all stages of CL development, and FGF22 protein was also detected in luteal tissue. FGF22 mRNA expression was lower at stage IV than at stage III (P < 0.05), and the same pattern was observed in luteal immunoreactivity. Furthermore, cloprostenol-induced luteolysis, both total and partial, increased FGFR1B mRNA abundance in luteal tissue (P < 0.05), but did not affect FGF22 mRNA abundance. In conclusion, these data suggest a potential role for the FGF22-FGFR1B system during development and regression of bovine CL.

Plasma pgfm and progesterone concentrations, luteolysis moment and estrous cycle length in Nelore cows submitted to uterine biopsies / Concentrações plasmáticas de pgfm e progesterona, luteólise e comprimento do ciclo estral em vacas Nelore submetidas à coleta de fragmentos endometriais

In this study, endometrial samples were collected in 14 Nelore cows on days zero (ovulation), five, nine, thirteen and nineteen of the estrous cycle (biopsy group), and in 15 females these collections weren't performed (control group). Biopsies were done on the uterine horn endometrium contralateral to the ovary with corpus luteum. Blood samples were taken at -24, -16, -8, 0 +8, +16 and +24 hours in relation to progesterone drop (<1ng/mL, zero moment) and evaluated for 13, 14-dihydro-15-keto prostaglandin F2-alpha (PGFM) by radioimmunoassay (RIA). Plasma progesterone concentration was determined by RIA every 24 hours. Data were analyzed by ANOVA using the PROC GLM and MIXED of the SAS. The mean value for PGFM during the entire period evaluated was greater in the biopsy group. The mean concentration of PGFM at moment zero was not different between the groups; the mean concentration of PGFM was higher in the biopsy group before and after the drop in progesterone level. The maximum mean concentration observed was not different between the groups; however, the mean minimum concentration was different with high values in the biopsy group. Although the PGFM concentrations were higher in the biopsy group, the biopsy and control groups had similar length of estrous cycle showing that repeated endometrial biopsy on the side contralateral to the ovary with corpus luteum does not affect luteolysis and the length of the estrous cycle.

No presente estudo, foram coletadas amostras endometriais de 14 vacas Nelore nos dias zero (dia da ovulação), cinco, nove, 13 e 19 do ciclo estral (grupo controle), e em 15 fêmeas essas coletas não foram realizadas (grupo controle). As biópsias foram realizadas no corno uterino contralateral ao ovário contendo o corpo lúteo. Amostras plasmáticas foram coletadas nos momentos -24, -16, -8, 0 +8, +16 e +24 horas em relação à queda de progesterona (<1ng/mL, momento zero) e avaliadas quanto à concentração de 13, 14-di-hidro-15-ceto prostaglandina F2-alpha (PGFM) por radioimunoensaio (RIA). As concentrações plasmáticas de progesterona foram avaliadas a cada 24 horas também por RIA. Os dados foram analisados por ANOVA empregando-se PROC GLM e MIXED do SAS. O valor médio de PGFM durante todo o período avaliado foi maior no grupo biópsia. A concentração média de PGFM no momento zero não diferiu entre os grupos, e foi maior no grupo biópsia antes e após a queda de progesterona. A concentração máxima observada não foi diferente entre os grupos, porém a concentração mínima diferiu com maiores valores observados no grupo biópsia. Embora as concentrações de PGFM fossem maior no grupo biópsia, ambos os grupos apresentaram o mesmo comprimento do ciclo estral, demonstrando que a coleta repetitiva de biópsias endometriais no corno uterino contraletral ao ovário contendo o corpo lúteo não afeta a luteólise e o comprimento do ciclo estral.

Fetal malformation in a marmoset (Callithrix jacchus): case report / Malformação fetal em sagui-de-tufo-branco (Callithrix jacchus): relato de caso

Callithrix jacchus is a neotropical primate adaptable in captivity. Colonies can be easily established in a short time and at low cost compared to other species of larger primates, which are normally used in laboratory. Because they are phylogenetically similar to humans in situations that induce anxiety, these small primates are increasingly being used in research involving the stress response. Wild animals in captivity are subjected to a series of stressful events that depending on the duration and intensity can modify the organic homeostasis. Observed in this study, serious problems occurred with the formation in fetal offspring of Callithrix jacchus kept in an environment with a high degree of stress...

O Callithrix jacchus é um primata neotropical de fácil adaptação em cativeiro. As colônias podem ser facilmente estabelecidas em um curto espaço de tempo e a um custo baixo comparado a outras espécies de primatas maiores, que são normalmente utilizados em laboratório. Por serem filogeneticamente semelhantes aos seres humanos em situações que induzem à ansiedade, esses pequenos primatas estão sendo cada vez mais utilizados em pesquisas que envolvam a resposta ao estresse. Animais silvestres em cativeiro estão submetidos a uma série de eventos estressores, os quais, dependendo da sua duração e intensidade, podem modificar a homeostase orgânica. Observaram-se, neste estudo, graves problemas ocorridos com a formação fetal nas proles de Callithrix jacchus mantidos em ambiente com alto grau de estresse...

Detecção imunoistoquímica de receptores de estrógeno e progesterona no endométrio de vacas Nelore (Bos taurus indicus) durante o anestro pós-parto / Immunohistochemical detection of receptors for estrogen and progesterone in endometrial glands and stroma during the pospartum anestrus in Nelore (Bos taurus indicus) cows

Foram utilizadas 24 vacas Nelore P.O., em anestro pós-parto, diagnosticado pelo histórico reprodutivo, por avaliações ultrassonográficas transretais e por dosagem de progesterona plasmática, que foram submetidas à colheita de fragmento uterino via transcervical. Os animais foram divididos em dois grupos conforme o máximo diâmetro folicular: grupo 1: folículos <6mm (n=12); grupo 2: folículos >6mm (n=12). Para avaliar receptor de estrógeno e receptor de progesterona no epitélio glandular e no estroma, foi utilizada a técnica de imunoistoquímica. Altas contagens relativas e alta intensidade de marcação para receptor de estrógeno e progesterona no epitélio glandular e no estroma foram observadas nos dois grupos. No entanto, a intensidade de marcação para o receptor de progesterona no epitélio glandular foi mais alta no grupo 2 comparado ao grupo 1. Quando o epitélio glandular e o estroma foram comparados, o número relativo de receptor de estrógeno no grupo 1 foi mais alto no epitélio glandular comparado ao estroma, e a intensidade de marcação para o receptor de progesterona no grupo 2 foi mais alta no epitélio glandular comparado ao estroma. Os resultados sugerem que os mecanismos que controlam a expressão de receptores no anestro são semelhantes aos observados durante o ciclo estral.

Twenty-four postpartum anestrous Nelore purebred cows were used in the study. Anestrous was determined based on the reproductive history which was confirmed in each cow based on plasma progesterone concentration and by transrectal ultrasonography. Endometrial biopsies were collected. The animals were separated into two groups according to maximum follicular diameter- Group 1: follicles <6mm (n=12) and Group 2: follicles >-6mm follicles (n=12). The immunohistochemistry technique was employed to evaluate the presence of estrogen and progesterone receptors in the uterine glandular epithelium and stroma. High counts of positive nuclei and high intensity of immunostain for estrogen and progesterone receptors in the glandular epithelium and stroma were observed in the two groups. However, the immunostain intensity of progesterone receptors in the glandular epithelium was higher in Group 2 compared to Group 1. When glandular epithelium and stroma were compared within each group, the relative number of estrogen receptors in the Group 1 was higher in the glandular epithelium compared to stroma and the immunostain intensity for the progesterone receptor in Group 2 was higher in the glandular epithelium compared to stroma. The results suggest that the mechanisms that control the expression of endomerial receptors in the anestrus are similar to the ones observed during the estrus cycle.

Impact of 24-h cooling prior to freezing on the survival of domestic cat (Felis catus) epididymal sperm.

The aim of this study was to investigate the impact of a 24-h cooling period prior to freezing on domestic cat epididymal sperm viability. Fifteen tomcats were submitted to routine orchiectomy and sperm samples were retrieved from both epididymides in a Tris-glucose-20% egg yolk extender. For each tomcat, the diluted sperm was split into two equal volumes and cooled to 5 degrees C at a rate of 0.5 degrees C/min; one sample for 60 min (control) and the other for 24 h (cooled). After the cooling period, samples from both groups were frozen using an identical freezing protocol. Sperm samples were evaluated in three different periods: immediately after harvesting, after cooling at 5 degrees C for 24 h (cooled group) and after freezing-thawing of control and cooled groups. Evaluations consisted of sperm motility and progressive status, sperm morphology and plasma membrane integrity (PMI) using two fluorescent probes. After cooling for 24 h, a decrease (p < 0.05) in sperm motility, progressive status and PMI was observed when compared to sperm samples immediately after collection. Comparing the results obtained after thawing, no difference (p < 0.05) was found regarding sperm motility, progressive status, PMI and sperm morphology between control and cooled groups. The results from the present study show that cooling cat epididymal spermatozoa at 5 degrees C for 24 h prior to freezing does not lead to major damage of spermatozoa impairing the freeze-thaw process.

Immunohistochemical detection of receptors for oestrogen and progesterone in endometrial glands and stroma during the oestrous cycle in Nelore (Bos taurus indicus) cows.

The aim of the present study was to monitor endometrial distribution and concentrations of oestrogen receptors alpha (ER alpha) and progesterone receptors (PR) by immunohistochemistry in Nelore cows (Bos taurus indicus) during the oestrous cycle. Blood samples were collected for progesterone measurement and endometrial samples were taken from the uterine horn contra lateral to the corpus luteum in 16 cows at days 0 (ovulation), 5, 9, 13 and 19 of the oestrous cycle. Immunostaining evaluation for ER alpha and PR in the glandular epithelium and uterine stroma was performed by two methods: positive nuclei counting and staining intensity of the nuclei. Specific positive staining reactions for both receptors were limited to cell nuclei and they were not identified in the cytoplasm. The proportion of ER alpha positive nuclei had a temporal variation throughout the oestrous cycle in both cell types evaluated and was higher in uterine stroma than the glandular epithelium (p < 0.05). The greatest proportion of ER alpha stained nuclei was observed at oestrus and during the initial and mid luteal phase (days 5, 9 and 13) (p < 0.05) in the glandular epithelium and at days 0, 5 and 9 in the uterine stroma (p < 0.01). The proportion of PR positive nuclei remained constant throughout the entire oestrous cycle for both cell types evaluated (p > 0.05). A higher proportion of PR positive nuclei was measured in the uterine stroma compared with the glandular epithelium (p < 0.05). Intensity of staining for ER alpha and PR varied throughout the oestrous cycle (p < 0.01). There was a higher staining intensity at days 0 and 5 in the stroma for ER alpha (p < 0.01) and PR (p < 0.01) and in the glandular epithelium at days 0, 5, 9 and 13 for ER alpha (p < 0.01) and at days 0, 5 and 9 for PR (p < 0.01) when compared with the other evaluated days. These data demonstrate that ER alpha and PR expression varied throughout the oestrous cycle in Nelore cows, in general with highest concentrations at oestrus and the lowest during the luteal phase. This is similar to patterns observed in Bos taurus taurus.

Diagnóstico ultra-sonográfico de piometra em cadelas / Ultrasonographic diagnosis of pyometera in bitches

A ultra-sonografia do tipo B (SCANNER 450 (5MHz), Pie Medical, Holanda) foi utilizada isolada ou em conjunto com exames laboratoriais e radiográficos em 33 cadelas com história clínica compatível com piometra. O útero dilatado apresentou imagem ultra-sonográfica de uma estrutura tubular bem definida com diâmetro entre 0,5 e 4,0 cm. O conteúdo luminal uterino apresentou menor ecogenicidade que a parede, com cintilaçöes ecogênicas bem evidentes. Houve concordância entre o aumento da viscosidade da secreçäo e a intensidade ecogênica. O diagnóstico ultra-sonográfico foi possível em 31 animais (94 por cento), e foi confirmado através de laparotomia ou necrópsia. Concluímos que a ultra-sonografia do tipo B é método eficiente, podendo ser utilizado no diagnóstico da piometra canina