Infertility associated with the absence of endometrial progesterone receptors in a bitch

A three‐year‐old intact female Old English sheepdog was presented for evaluation of infertility. A uterine biopsy was performed during dioestrus, and the microscopic appearance was inconsistent with progesterone stimulation; the glands were sparse, simple and failed to show coiling, while the glandular epithelium was cuboidal instead of columnar. There was very little evidence of glandular activity. Due to the inappropriate appearance of the glands for the stage of the cycle, immunohistochemistry for progesterone receptors was performed. No progesterone receptor‐positive immunoreactivity was identified in the endometrial luminal epithelium, glandular epithelium or stroma. Weak intranuclear immunoreactivity was identified within the smooth muscle cells of the myometrium. The absence of progesterone receptors within the endometrial glands is the most likely explanation for the abnormal appearance of the endometrium and for this bitch's infertility. To our knowledge, this is the first report of endometrial progesterone receptor absence in a bitch.     

Evaluating the behaviour of vertical structure indices in Scots pine forests

? Stand structure indices would appear to be good surrogate measures for biodiversity in forest ecosystems.

? The vertical structure of Pinus sylvestris L. stands in Central Spain was analysed in order to evaluate their structural diversity. A comparison between two forests with similar ecological conditions but managed under different silvicultural systems was conducted in order to analyse changes in diversity at different stages of stand development. Height diversity was quantified using two non-spatially explicit indices (Shannon’s index and STVI) as well as two spatially explicit indices (Gadow’s differentiation index and the Structure complexity index). A new diversity index was then proposed, based on the sum of square roots of height differences (SQRI).

? Correlations between all vertical structure indices were highly significant. All indices showed that height diversity was greater in the forest with the longer regeneration period and where less intensive thinnings were applied throughout the rotation. Diversity was highest in uneven-aged stands and in the period between the regeneration stage and the first thinning. Thinning from below accounted for the decrease in vertical structure complexity throughout the rotation in even-aged stands.

? The results show that height distribution along with successional stage data enhance the analysis of vertical diversity since structural complexity is highly related to the silvicultural practices that are carried out at different ages.

     

Effects of Solanum glaucophyllum toxicity on cell proliferation and apoptosis in the small and large intestine of rabbits

Vitamin D regulates mineral homeostases and enterocyte proliferation and differentiation. Hypervitaminosis D generates changes in cell proliferation, differentiation and apoptosis in several organs. We analysed morphometric parameters and proliferative and apoptotic indices in the intestinal epithelium of rabbits with hypervitaminosis D induced by the chronic treatment with the calcinogenic plant Solanum glaucophyllum. Rabbits were treated for 15 or 30 days. A group was treated for 15 days and led to possible recovery for 30 days. Another group was nutritionally restricted for 30 days. Morphological, morphometric, proliferative and apoptotic changes were found in the treated animals. Mild atrophy and reduced proliferation was found in the jejunum and ileum. Apoptosis increased in the crypts of the ileum and in the superficial epithelium and crypts of the rectum. Most of the alterations were partially recovered. The possible involvement in these changes of the hypervitaminosis D-like state induced by S. glaucophyllum is discussed.     

Postnatal exposure to a progestin does not prevent uterine adenogenesis in domestic dogs

To assess the effects of a single supraphysiological postnatal administration of a progestogen on uterine glands in dogs, 10 females were randomly assigned to a medroxyprogesterone acetate 35 mg (MPA; n = 6) or placebo (n = 4) group within the first 24 h of birth. The safety of the treatment was also evaluated. A transient mild clitoris enlargement appeared in MPA-treated females. Microscopic postpubertal uterine assessment revealed the presence of uterine glands in all cases without significant differences in the area occupied by the glands per µm² of endometrium nor in the height of the uterine epithelium.     

Insulin-like growth factor I in sera, ovarian follicles and follicular fluid of cows with spontaneous or induced cystic ovarian disease

The objective of this research was to determine changes in IGF-I levels in serum and follicular fluid, and immunoreactivity of the follicle wall of cows with spontaneous (slaughter specimens) or ACTH-induced follicular cysts, and to compare results to normal cycling (control) cows after selection of the ovulatory follicle. Concentrations of IGF-I in serum did not differ between control and cystic animals (p=0.76). Fluid from the ovulatory follicle in control cows had 41% higher concentrations of IGF-I than that from cystic follicles collected at slaughter (spontaneous cysts; p<0.05) and 70% higher than that in induced follicular cysts (p<0.05). An intense positive immunostaining with anti-IGF-I was observed in granulosa cells (p<0.05) and in the theca interna (p<0.05) of secondary and tertiary follicles in all three groups of animals, but staining was less intense in cystic (p<0.05) and atretic follicles (p<0.05). This study provides evidence to suggest that cystic ovarian disease in cattle is associated with decreased concentrations of IGF-I in follicular fluid, but not in serum, and decreased production of IGF-I in the follicular wall. These data support the notion that IGF-I plays a role in the regulation of folliculogenesis, and may participate in the pathogenesis of cystic ovarian disease in cattle.     

Hepatic steroid inactivating enzymes,hepatic portal blood flow and corpus luteum blood perfusion in cattle

Production from the corpus luteum (CL) and/or hepatic steroid inactivation impacts peripheral concentrations of P4, which can alter reproductive performance. Our primary objective was to examine hepatic steroid inactivating enzymes, portal blood flow, and luteal blood perfusion at 10 days post‐insemination in pregnant versus non‐pregnant beef and dairy cows. Twenty early lactation Holstein cows and 20 lactating commercial beef cows were utilized for this study. At day 10 post‐insemination, hepatic portal blood flow and CL blood perfusion were measured via Doppler ultrasonography. Liver biopsies were collected and frozen for later determination of cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A), uridine diphosphate‐glucuronosyltransferase (UGT) and aldo‐keto reductase 1C (AKR1C) activities. Pregnancy was determined at day 30 post‐insemination and treatment groups were retrospectively assigned as pregnant or non‐pregnant. Data were analyzed using the mixed procedure of SAS. Steroid metabolizing enzyme activity was not different (p > .10) between pregnant versus non‐pregnant beef or dairy cows. Hepatic portal blood flow tended (p < .10) to be increased in pregnant versus non‐pregnant dairy cows. Luteal blood perfusion was increased (p < .05) in pregnant versus non‐pregnant dairy cows. Pregnant dairy cows appear to have an increased rate of hepatic clearance of P4 in combination with increased synthesis from the CL. This could account for the lack of difference in peripheral P4 concentrations between pregnant and non‐pregnant dairy cows. This study highlights the relevance of further investigation into steroid secretion and inactivation and their impact on the maintenance of pregnancy in cattle.     

Histochemistry of the zona pellucida of the ovary of a species with natural polyovulation: Lagostomus maximus (Rodentia,Hystricomorpha, Chinchillidae)

This study reports the histochemistry and the distribution of glycoconjugates (GCs) in the zona pellucida (ZP) of preantral, secondary, tertiary, polyovulatory and atretic follicles of ovaries from non‐pregnant (NPr) and pregnant (Pr) females of Lagostomus maximus. GCs were studied using histochemical and lectin histochemical methods. The viscacha ZP was positive to all the histochemical techniques. In addition, it was observed that the intensity of staining of the ZP was constant in the different follicular stages between both female groups. The lectin histochemical study revealed that ZP was positive for certain lectins (WGA, RCA‐I and CON‐A) and that the labelling did not vary between the different follicular stages, but between the two groups of females. By using both histochemical techniques, it was established that the GCs present in the ZP label the complexity of the area. These results allow us to increase our knowledge on the biology of the viscacha's ovary, particularly contributing to the study of polyovulation.     

Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre‐exposure of oocytes to Ricinus communis (RCA‐1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm–egg binding and fertilization. In vitro‐matured bovine oocytes were incubated (39°C, 5% CO₂ in air) for 2 h in the following treatments: (i) 500 μl of fertilization medium (FM); (ii) 250 μl of FM with 0.25 ml of non‐luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 μl of FM with 250 μl of NLAUTF and 4 μl of RCA‐1 lectin; (iv) 250 μl of FM with 250 μl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA‐1 lectin; (v) 500 μl of FM and RCA‐1 lectin. Following incubation, oocytes were washed, placed in FM with 2 μg heparin, and incubated with 1 × 10⁵ frozen–thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate‐orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean ± SEM) when oocytes were incubated in treatment 3 (59.0 ± 5.5) than in treatments 2 (46.4 ± 5.6), 4 (18.1 ± 5.4), 5 (33.4 ± 5.6) or 1 (32.5 ± 5.6). More oocytes were fertilized when incubated in treatment 3 (91% ± 3.0) than in 2 (84% ± 3.0), 4 (40% ± 3.0), 5 (77% ± 3.0) or 1 (76% ± 3.0). As in previous studies, this study suggests that RCA‐1 lectin enhances binding of UTF‐derived OPN to bovine oocytes, resulting in increased sperm–egg binding and fertilization in vitro and a possible role in fertilization.     

Ovine rumen papillae biopsy via oral endoscopy; a rapid and repeatable method for serial sampling

AIMS: To explore and validate the utility of rumen endoscopy for collection of rumen papillae for gene expression measurement.

METHODS: Four adult Coopworth ewes were fasted for either 4 or 24 hours. Animals were sedated, placed in a dorsally recumbent position at 45 degrees with the head upright, and an endoscope inserted via a tube inserted into the mouth. Biopsies of rumen papillae were taken from the ventral surface of the rumen atrium under visual guidance. Two biopsies were collected from one of the animals that had been fasted for 4 hours, and three from one of the animals that had been fasted for 24 hours. Video of the rumen atrium and reticulum was also collected. The animals recovered uneventfully. Biopsies were subsequently used for extraction and sequencing of mRNA.

RESULTS: The ventral surface of the rumen atrium was accessible after 4 hours off pasture, but a larger region was accessible after 24 hours of fasting. Sedation allowed access for endoscope use for around 5 to 10 minutes after which increased saliva flow was noted. Rumen papillae biopsies were easily collected, with samples from a variety of sites collected in the ～10 minute time window. High quality RNA was obtained for stranded mRNA sequencing. Of the resulting reads, 69–70% mapped uniquely to version 3.1 of the ovine genome, and 48–49% to a known gene. The rumen mRNA profiles were consistent with a previously reported study.

CONCLUSIONS: This method for obtaining rumenal tissue was found to be rapid and resulted in no apparent short or long term effects on the animal. High quality RNA was successfully extracted and amplified from the rumen papillae biopsies, indicating that this technique could be used for future gene expression studies. The use of rumen endoscopy could be extended to collection of a variety of rumen and reticulum anatomical measurements and deposition and retrieval of small sensors from the rumen. Rumen endoscopy offers an attractive and cost effective approach to repeated rumen biopsies compared with serial slaughter or use of cannulated animals.