Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P₄). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25°C, were incubated for 30 min at 38°C in 5% CO₂ and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P₄ was added (10 μg/ml) to the P1 group. Groups P2 and P3 were supplemented with P₄ (10 and 20 μg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 μ m , respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P₄ treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P₄ groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 μ m seems to be more suitable to trigger AR in domestic cat sperm.     

Articular / epiphyseal osteochondrosis in Thoroughbred foals at 5 months of age: influences of growth of the foal and prenatal copper supplementation of the dam

AIMS: To determine the influence of copper (Cu) supplementation by injection of mares in late gestation on the frequency and severity of osteochondrosis (OC) lesions in their foals at around 160 days of age. To determine if there was any influence of the concentration of Cu in the liver, growth rate, birthweight, weight at 160 days of age, fatness, sex, or year of birth of the foal on the frequency and severity of OC lesions. To determine the influence of dam's age, and sex and birthweight of the foal on the growth rate from birth to 160 days of age, and weight at 160 days of age. METHODS: Thirty-three Thoroughbred foals, born in two consecutive years, were weighed every 2 weeks from birth. The dams had been supplemented with parenteral Cu or saline during late gestation, and the supplementation regimens were different in each year. Foals had liver biopsies harvested at birth for determination of Cu concentration. Pasture samples were collected every 4-8 weeks for analysis of concentration of Cu and zinc (Zn). At 160 days of age, articular cartilage of long bones was examined. Gross lesions were counted and scored, then sawn and radiographed, and processed for histopathology. Lesions were given radiographic scores and histopathological scores. Maximum scores for each lesion were combined to give a total OC score for each joint and each foal. The fatness of 20 foals (10 each from Years 1 and 2) at 160 days of age was determined chemically. RESULTS: Supplementation of dams with Cu had no significant effect on the concentration of Cu in the liver of foals at birth, or on the frequency or severity of lesions in articular cartilage at 160 days of age. The Cu and Zn concentrations of pasture were similar in Years 1 and 2, and were lower than current recommendations. All foals in Year 2, and 9/10 foals in Year 1 had irregularities in cartilage that was confirmed histologically to be indicative of OC. The average number of lesions per foal was 4.7 (SD 1.1) and 5.7 (SD 1.1) in Years 1 and 2, respectively. However, the severity of the lesions was considered mild, and no foals showed any clinical evidence of OC while alive. The number of lesions in the tarsocrural (TC) joint and the TC OC score at 160 days were positively associated with average daily weight gain (ADG) in the previous 4 weeks (p=0.005 and p=0.001, respectively). There was no significant effect of sex, fatness, birthweight, weight at 160 days of age, or year of birth of the foal on the frequency and severity of OC lesions. CONCLUSIONS: Many of the lesions classified as OC, using classification systems described by other authors, were likely to be normal variations of the process of endochondral ossification. Despite the high frequency of such lesions, they were considered to be of minor significance and none were clinically evident. The distribution of lesions was not typical, and most probably reflected the subtlety of the lesions. These results support the hypothesis that Cu is an over-emphasised factor in the aetiopathogenesis of OC. The relationship between subtle macroscopic lesions and lesions resulting in clinical signs of disease requires further investigation.     

Physiological traits in preterm calves during their first week of life

Morbidity and mortality of preterm neonatal calves are higher than of calves born at normal term, possibly and in part due to immaturity of physiological functions. Physiological parameters were therefore studied during the first week of life in seven preterm calves, born on day 277 of gestation after dams were injected prostaglandin F2alpha and flumethason. Calves were fed colostrum of the first milking for the first 3 days and from day 4 to day 7 the same colostrum diluted with milk replacer. Body weight increased during the first week of life by 2.2 kg. Heart rate and respiratory rate were always relatively high, whereas values of rectal temperature, blood gases, haematological, metabolic and endocrine traits were in the range and behaved similarly as is the experience in full-term neonatal calves. Major exceptions were glucose and insulin, the concentrations of which barely rose postprandially, and growth hormone, the responses of which to growth hormone releasing factor analogue 1-29 were extremely variable and in part very small. In conclusion, calves born 2 week before normal term that survived the first week of life, although physiologically immature, were well able to handle ingested nutrients and to control their metabolism.     

α‐6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α‐6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self‐renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two‐step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α‐6 integrin by flow cytometry and real‐time RT‐PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two‐step enzymatic digestion. An average of 1 × 10⁵ viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α‐6 integrin expression. Flow cytometry analysis demonstrated no differences in the α‐6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real‐time PCR analysis (p > 0.05). In addition to α‐6 integrin, the expression of GFRa‐1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α‐6 integrin expression.     

The effect of individual liveweight and condition scores of ewes at mating on reproductive and scanning performance

AIMS: To determine the effects of liveweight (LW) and condition score (CS) of ewes at mating on both mating performance and scanning percentage, and to determine if effects of CS were independent of LW. METHODS: Two groups of ewes, comprising 1,780 mixed-aged Romney (Rom MA) and 692 composite two-tooth (1/8 East Friesian 1/16 Finn 13/16 Romney, Comp TT) ewes were grazed separately with harnessed rams for two reproductive cycles. Romney rams were used for the first 17 days of mating and then replaced on the morning of Day 18 by Suffolk rams. Ewes were weighed and CS assessed on a scale of 0-5 before the introduction of the rams. Ewes were identified as having been mated during the first cycle only, during both cycles, during the second cycle only, or not mated. All mated ewes were scanned using ultrasound, 50 days after the end of mating, and identified as being either non pregnant, single-, twin- or triplet-bearing. RESULTS: Linear relationships (R2=0.99) between mean LW and CS at mating were evident for both Rom MA and Comp TT ewes and the mean increase in LW per unit increase in CS was greater in the Rom MA ewes (7.88 kg) than the Comp TT (4.78 kg) ewes (p<0.05). Ewes mated in the second cycle only were significantly (p<0.05) lighter than those mated in the first cycle only. In Rom MA ewes, a greater proportion were mated in the first cycle only if CS was >/=2.0 than if CS was <2.0, and the proportions of ewes mated in the first cycle only or in both cycles did not differ if CS was >/=2.5. In Comp TT ewes, there was no difference in the proportions mated in the first cycle or both cycles, if CS was >/=2.0. Triplet-bearing ewes were heavier than twin-bearing ewes, which were in turn heavier than both singleton-bearing and non pregnant ewes. Twin-bearing ewes had higher CS than both single-bearing and non pregnant ewes (p<0.05) but differences were not significant if LW was included as a covariate. In both Rom MA and Comp TT ewes, greater proportions were non pregnant and lower proportions had twins if CS was <2.0 than if CS was >/=2.0. In Rom MA ewes only, a greater proportion again had twins and a lower proportion had singletons if CS was >/=3.0 than if CS was <3.0. In Comp TT ewes, the proportion that were non pregnant or twin-bearing did not vary with CS if CS was >/=2.0. Reproductive performance was maximal for Rom MA and Comp TT ewes that had CS at mating >/=3.0 and >/=2.0, respectively, equating to LW of 62.6 and 48.5 kg, respectively. CONCLUSIONS: In both groups of ewes, CS and LW at mating positively affected reproductive performance measured at the time of mating and at scanning, however, no reproductive advantage was evident above a minimum CS of 3.0 and 2.0, equating to LW of 62.6 and 48.5 kg, for Rom MA and Comp TT ewes, respectively. Sheep farmers should direct management to ensure that no ewes are below these minimum targets rather than ensuring high average LW and/or CS of a mob.     

Growth performance, metabolic and endocrine traits, and absorptive capacity in neonatal calves fed either colostrum or milk replacer at two levels

Colostrum (CO) contains high amounts, whereas whole milk and milk replacer (MR) contain small amounts, of bioactive and growth-promoting substances, such as IGF-I. An experiment was designed to study the effects of feeding CO or MR on the first 3 d to neonatal calves, followed by whole milk up to d 7, at low and high density. Intestinal absorptive capacity, plasma metabolite and hormone concentrations, and growth performance were measured during the 1st wk of life. Body weight increased (P < .05) similarly in calves fed low or high amounts of CO but did not rise in MR-fed calves. Loose feces were more frequent (P < .05) and absorption of xylose on d 5 was lower (P < .01) in MR- than in CO-fed calves, but there were no effects of feeding density within CO-fed or within MR-fed groups. However, high feeding density within CO-fed groups enhanced (P < .05) total protein, globulin, triglyceride, cholesterol, and insulin concentrations, whereas in the initially high and low MR-fed groups only plasma glucose and insulin after the first meal and plasma NEFA on d 2 were modified (P < .05) by different feeding density. Thus, feeding different amounts of CO partly influenced protein and fat metabolism in calves during the 1st wk of life, but it did not measurably affect intestinal function. However, feeding different amounts of MR, in the absence of CO, barely affected metabolic and endocrine traits and absorptive capacity. Thus, high density CO feeding, and therefore a high supply of nutrients, together with greater amounts of bioactive and growth-promoting substances influenced neonatal metabolism and growth more than a high density of MR feeding containing only small amounts of bioactive and growth-promoting substances. Factors in addition to nutrient density seem to be important for the development of neonatal calves.     

Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies

Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPAR, PPARγ) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione-S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n = 7 per group). Upon first presentation of dogs, mRNA levels of PPAR, PPARγ, CAR, PXR and RXR in duodenum as well as PPARγ, CAR, PXR and RXR in colon were not different among groups (P > 0.10). Although mRNA abundance of PPAR in colon of dogs with FRD was similar in both IBD and CON (P > 0.10), PPAR mRNA abundance was higher in IBD than CON (P < 0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD (P < 0.05) or CON (P < 0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON (P < 0.05). Differences in mRNA levels of PPAR and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD.