Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E₂), progesterone (P₄), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E₂-active (EA) and E₂-inactive (EI) follicles were selected according to their E₂-to-P₄ ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E₂ and P₄ concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.     

Alterations in coagulation parameters in dairy cows affected with acute mastitis caused by <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">S. aureus</Emphasis> pathogens

This study was conducted to evaluate alterations in coagulation parameters in dairy cows affected with acute Escherichia coli (E. coli) mastitis and to compare those values to cows affected with Staphylococcus aureus (S. aureus ) mastitis. Twenty-four, adult Holstein-Friesian dairy cows affected with acute E. coli mastitis and 17 cows affected with S. aureus mastitis were studied. Cows affected with E. coli mastitis had significantly prolonged activated partial thromboplastin time (APTT) (P < 0.01), prothrombin time (PT) (P < 0.05) and decreased (P < 0.05) platelets numbers. Cows with S. aureus mastitis had only significantly prolonged APTT (P < 0.05) and decreased (P < 0.05) platelet counts. In the hematology evaluation, cows affected with E. coli and those affected with S. aureus mastitis had elevated hematocrit values but only significantly (P < 0.05) so in mastitic cows caused by E. coli. Both groups of mastitic cows had significantly (P < 0.05) lower leukocyte counts. Only cows with E. coli mastitis had significantly (P < 0.05) lower neutrophil count. In the plasma biochemical evaluation, creatinine concentrations were significantly (P < 0.05) elevated in both groups of cows. Blood urea nitrogen (BUN) concentration was only significantly elevated in cows affected with E. coli mastitis. Results of this study indicated that dairy cows affected with acute E. coli mastitis are more likely to develop clinical manifestations of disseminated intravascular coagulation than cows affected with S. aureus mastitis.     

Predictive value of prepartum serum metabolites for incidence of clinical and subclinical mastitis in grazing primiparous Holstein cows

The aim of this study was to measure changes in biochemical markers in the peripartum period of primiparous Holstein cows diagnosed with subclinical and clinical mastitis. In this study, 37 dairy cows were monitored daily during milking until 60 days postpartum and were categorized according to the occurrence of clinical mastitis (group mastitis (GM), n?=?9) or subclinical mastitis (group subclinical mastitis (GSUB), n?=?10) or absence of symptoms (control group (CG), n?=?18). Blood samples were collected weekly from ?30 to 60 days from calving. Samples were grouped for prepartum (?30 to 0 days from calving), early postpartum (0 to 30 days from calving), and late postpartum (30 to 60 days from calving) periods. Prepartum serum non-esterified fatty acid (NEFA) concentration was higher in GM than in CG (P?<?0.01). In addition, CG had higher prepartum serum glucose concentration than GM (P?=?0.03). In the early postpartum period, aspartate aminotransferase (AST) activity was lower in CG than in GSUB (P?<?0.05), and in the late postpartum period, AST activity was lower in CG than GSUB and GM (P?=?0.01). Somatic cell count was higher during the early and late postpartum periods for GM and GSUB when compared to CG (P?<?0.01). In this study, primiparous cows with low glucose and higher NEFA in the prepartum were more susceptible for mastitis in the early postpartum, probably due to low immune function associated to a more negative energy balance. In sum, increased prepartum serum NEFA concentration and decreased glucose in primiparous cows were associated with clinical mastitis incidence in the postpartum period.     

Ovarian localization of 11β-hydroxysteroid dehydrogenase (11βHSD): effects of ACTH stimulation and its relationship with bovine cystic ovarian disease

Cystic ovarian disease (COD) is an important cause of infertility in cattle, and ACTH has been involved in regulatory mechanisms related to ovarian function associated with ovulation, steroidogenesis, and luteal function. Here, we examined the localization of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and 11βHSD2 proteins in the ovary of healthy cows and animals with spontaneous and ACTH-induced COD and the in vitro response of the follicular wall exposed to ACTH. After stimulation by ACTH, we documented changes in 11βHSD expression and cortisol secretion by the follicular wall of large antral and follicular cysts. Follicular cysts showed a higher constitutive expression of both enzymes, whereas ACTH induced an increase in 11βHSD1 in tertiary follicles and follicular cysts and a decrease in 11βHSD2 in follicular cysts. Moderate expression of 11βHSD1 was observed by immunohistochemistry in granulosa of control animals, with an increase (P < 0.05) from primary to secondary, tertiary, and atretic follicles. The level of immunostaining in theca interna was lower than that in granulosa. The expression of 11βHSD2 was lower in the granulosa of primary follicles than in that of secondary, tertiary, and atretic follicles and was lower in the theca interna than in the granulosa. In ACTH-induced and spontaneously occurring follicular cysts, differences from controls were observed only in the expression of 11βHSD1 in the granulosa, being higher (P < 0.05) than in tertiary follicles. These findings indicate that follicular cysts may be exposed to high local concentrations of active glucocorticoids and indicate a local role for cortisol in COD pathogenesis and in regulatory mechanisms of ovarian function.     

Subclinical mastitis causes alterations in nitric oxide, total oxidant and antioxidant capacity in cow milk

The aim of this study was to investigate total antioxidant (TAC), and oxidant capacity (TOC) and nitric oxide (NO) levels in milk of cows with subclinical mastitis. Brown Swiss and Holstein breed cows were screened with California Mastitis Test (CMT) to determine mammary glands with subclinical mastitis. Moreover, somatic cell counts (SCC) were determined electronically in all milk samples. Mammary quarters were classified as healthy (n = 25) or subclinical mastitis (n = 35) based on CMT scores and somatic cell count (SCC: ?200,000/ml or >200,000/ml) in milk. Nitric oxide, TOC and SCC levels were significantly higher (p < 0.001, p < 0.005 and p < 0.001, respectively) in milk from mammary quarters with subclinical mastitis compared to those from healthy mammary quarters. In conclusion, subclinical mastitis results in higher NO concentrations, TOC and SCC, and NO and TOC were positively correlated with SCC. Moreover, alterations in NO levels and TOC in milk could be used as an alternative diagnostic tool to screen for subclinical mastitis.     

Tumor necrosis factor-alpha (TNF alpha) inhibits progesterone and estradiol-17beta production from cultured granulosa cells: presence of TNFalpha receptors in bovine granulosa and theca cells

The aim of this study was to investigate whether functional tumor necrosis factor-alpha (TNFalpha) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P(4)) and estradiol-17beta (E(2)) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 +/- 0.15 to 6.9 +/- 1.40 nM). Moreover, TNFalpha receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFalpha (rhTNFalpha; 0.06-6 nM) inhibited E(2) secretion in a dose-dependent fashion (P<0.01), but did not affect P(4) secretion. In addition, rhTNFalpha inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P(4) and E(2) secretion by the cells (P<0.01). These results indicate the presence of functional TNFalpha receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFalpha plays a role in regulating their secretory function.     

Relationships among vasculature,mitotic activity,and endothelial nitric oxide synthase (eNOS) in bovine antral follicles of the first follicular wave

To determine the relationships among vasculature, mitotic activity, and expression of endothelial nitric oxide synthase (eNOS) of antral follicles in Bos indicus, bovine ovaries were obtained on day 6 of the estrous cycle from 10 crossbred (Brahman to Thai native cows) after a synchronized estrus with prostaglandin F₂α analogue. Ovaries were fixed, paraffin-embedded, and used for immunofluorescence detection of factor VIII (a marker of endothelial cells). Immunostaining of eNOS and proliferating cell nuclear antigen (PCNA) were performed with specific monoclonal antibodies. Vasculature and positive staining of eNOS and PCNA were quantitatively evaluated with the image analysis. Follicles were classified by size (small, medium, and large) and by structure as healthy and atretic follicles (n = 82). The expression of factor VIII and eNOS were detected greater in the blood vessels of the theca layers of the healthy follicles than those in atretic follicles. The labeling indices (LIs) in granulosa and theca cells were greater (P < 0.05) in the healthy small and medium follicles than in the healthy large follicles. Vasculature, capillary area density, and capillary number density were positively correlated with eNOS expression and the LIs of granulosa and theca cells but were negatively correlated with the healthy follicle size. During the growing phase of antral follicle in Bos indicus, relationships among vasculature, mitotic activity, and eNOS were observed predominantly in healthy antral follicles. Thus, these data highlight the importance of vasculature, cell proliferation, and eNOS expression of growing and atretic follicles in the first follicular wave.     

Udder health in relation to udder and teat morphometry in Holstein Friesian × Sahiwal crossbred dairy cows

The present investigation involved 261 Holstein Friesian (HF) × Sahiwal cows to study the udder health as well as the udder and teat morphometry. The udder health was defined on the basis of bacteriology and California mastitis test of quarter foremilk. The morphometry parameters included udder fore depth (UFD, distance from the point where the fore udder merges within the abdomen to a point in front of the fore teats at the level of the udder base), udder rear depth (URD, distance from the bottom of the vulva to the base of the rear udder), udder depth (UD, level of the udder base with respect to hock joint), height of the udder from the floor, teat length, teat diameter (at mid of the teat barrel), teat-tip to floor distance and distance between the teats. The URD, UD and height of udder from the floor showed a significant (P?<?0.05) relation to the udder health with mastitic cows having larger URD, smaller udder distance from the floor and the udder base placed below the hock joint. The cows with teat length larger than 4.5 cm and teat diameter 3.0 cm or larger had significantly more quarters affected with mastitis. Further, cows with teats placed nearer to the floor were more susceptible to mastitis (P?<?0.01). No significant differences were found between healthy and mastitic udders with respect to UFD and distance between teats. It can be concluded that selection for optimum udder and teat morphometry in breeding programmes may help to reduce susceptibility to intramammary infections in HF × Sahiwal cows.     

Correlations among antral follicular echotexture,apoptosis and expression of key steroidogenic enzymes in sheep

Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3β-hydroxysteroid dehydrogenase, 3β-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3β-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays.     

Production of lipid mediators in mastitic milk of cow

Bovine mastitis is one of the most prevalent and costly diseases in the dairy industry. Lipid mediators are signaling molecules which coordinately and intricately modulate inflammation. They are produced from polyunsaturated fatty acids (PUFAs) in the cellular membrane via several enzymes including cyclooxygenase (COX) and lipoxygenase (LOX). In the present study, we performed comprehensive analysis of lipid production in milk obtained from clinical or subclinical mastitic cows using liquid chromatography/mass spectrometry. We detected 26, 24, and 40 kinds of lipid constantly in healthy, subclinical, and clinical mastitic milk, respectively. In clinical mastitic milk, the amount of a major n‐6 PUFA, arachidonic acid (AA), tended to increase, whereas amounts of major n‐3 PUFAs, eicosapentaenoic acid and docosahexaenoic acid, tended to decrease. The amounts of several AA‐derived lipids including COX‐catalyzed prostaglandin (PG) D₂ and PGE₂, and LOX‐catalyzed leukotriene (LT) B₄ were increased in clinical mastitic milk. Although subclinical mastitic milk represented similar trend of lipid production to healthy milk, amounts of several lipids such as LTD₄, 14,15‐dihydroxyeicosatrienoic acid, and 14‐epoxyeicosatrienoic acid changed. These findings would be helpful for better understanding of mastitis pathology and give us some insights to develop a new diagnostic and therapeutic strategy.     

Increased abundance of aromatase and follicle stimulating hormone receptor mRNA and decreased insulin-like growth factor-2 receptor mRNA in small ovarian follicles of cattle selected for twin births

Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF-1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives were to 1) assess relationships among aromatase (CYP19A1), IGF-1 (IGF1), IGF-2 receptor (IGF2R), and FSH receptor (FSHR) mRNA expression in small (≤5 mm) antral follicles and 2) determine their association with increased numbers of developing follicles in ovaries of Twinner females. Ovaries were collected from mature, cyclic (d 3 to 6) Twinner (n = 11), and Control (n = 12) cows at slaughter and pieces of cortical tissue were fixed and embedded in paraffin. Expression of mRNA was evaluated by in situ hybridization using (35)S-UTP-labeled antisense and sense probes for CYP19A1, FSHR, IGF1, and IGF2R mRNA. Silver grain density was quantified within the granulosa and theca cells of individual follicles (2 to 7 follicles/cow) by Bioquant image analysis. Follicles of Twinners tended to be smaller in diameter than Controls (1.9 ± 0.1 vs. 2.3 ± 0.1 mm; P = 0.08), but thickness of granulosa layer did not differ (P > 0.1) by genotype. Relative abundance of CYP19A1 (P < 0.01) and FSHR (P < 0.05) mRNA was greater in granulosa cells of Twinners vs. Controls, respectively, whereas IGF2R mRNA expression was less in both granulosa (P < 0.01) and theca (P < 0.05) cells in follicles of Twinners vs. Controls, respectively. Abundance of CYP19A1 mRNA in granulosa cells was correlated negatively with IGF2R mRNA expression in both granulosa (r = -0.33; P < 0.01) and theca (r = -0.21; P = 0.05) cells. Expression of IGF1 mRNA was primarily in granulosa cells, including cumulus cells, and its expression did not differ between Twinners vs. Controls (P > 0.10). Detected increases in CYP19A1 and FSHR, but not IGF1, mRNA expression along with decreases in IGF2R mRNA expression in individual follicles of Twinners support the hypothesis that increased follicular development and steroidogenesis in Twinner females result from increased extra-ovarian IGF-1 production. Furthermore, a reduction in follicular IGF2R mRNA expression accompanied by a reduction in receptor numbers would increase availability of free IGF-2 and its stimulation of follicular development in Twinners.     

Effect of oral administration of colostrum on inflammation in the udders of dairy cows suffering from mastitis

The present study was undertaken to examine whether oral administration of colostrum to mastitic cows reduced inflammation in the udder. Fifty milliliters of a colostrum whey product was administered orally daily for 3 days to cows suffering from mastitis. Milk was collected on day 0 and 7 of colostrum administration. For Experiment 1, milk from 11 udder quarters with high somatic cell counts (SCC) in four cows was used. SCC in milk decreased significantly after colostrum administration, whereas colostrum administration increased sodium and IgA concentrations significantly compared with those before administration. In Experiment 2, cows with clinical mastitis were divided into two groups, with and without colostrum administration, whereas all cows with subclinical mastitis were administered colostrum. Antibiotics were infused into the mammary gland from the first day of colostrum administration for 2–4 days. There was no significant decrease in SCC after colostrum administration in any group. However, udder firmness in both clinical mastitis groups was reduced after administration regardless of colostrum administration. IgA concentration in both clinical mastitis groups was significantly increased after colostrum administration compared to that before administration, although there was no significant difference between them. These results suggest the possibility that oral administration of colostrum attenuates inflammation of the mammary gland. Further studies are required to examine the effect of colostrum more precisely using cows with subclinical and chronic mastitis and longer duration of colostrum administration.     

Effects of the phytoestrogen,genistein, and protein tyrosine kinase inhibitor–dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles

The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P₄) and estradiol (E₂) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and β (ERα and ERβ) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)–dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P₄ secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P₄ secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E₂. In contrast, lavendustin C did not alter basal E₂ secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERβ (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERβ. However, the genistein action on P₄ and E₂ production by granulosa cells harvested from medium follicles did not seem to be associated with PTK.     

Risks factors associated with subclinical mastitis in water buffaloes in Pakistan

The present study was carried to ascertain the association of various risk factors of mastitis in water buffaloes. The milk samples from buffaloes were collected and screened through California Mastitis Test for the presence of mastitis. In the present study, 15.2 % prevalence of subclinical mastitis was recorded both at the government (13.4 %) and private farms (15.5 %). The chi-square analysis showed significantly higher involvement of the right rear and front quarters. The analysis of variance technique showed significant difference in live body weight, milk yield, teat end to floor distance (P?<?0.001), udder depth, teat length, and teat diameter in mastitic and healthy buffaloes. The frequency analysis also revealed significant difference between various groups including lactation stage, teat and/or udder pathology, teat shape, and udder shape (P?<?0.001). The logistic regression analysis revealed significant positive association of mastitis with milk leakage, live body weight, milk yield, parity, calf suckling, pendulous udder, number of attendants at the farm, dirty hind legs, and udder depth.     

Effect of mastitis on macro-minerals of bovine milk and blood serum in Sudan

Milk and blood serum from clinically mastitis infected, subclinically mastitis infected and healthy Friesian cows (15 samples from each of 3 groups) were evaluated for macrominerals (sodium, potassium, calcium, magnesium and phosphorus). The milk from cows infected with subclinical mastitis revealed a significant decrease in potassium (P < 0.001) and a significant increase in sodium and phosphorus content (P < 0.01). Similarly, the milk from cows with the clinical form of the disease showed a significant increase in sodium (P < 0.001) and a significant decrease in potassium, magnesium (P < 0.001) and calcium (P < 0.01). Comparison of healthy cow's milk with that from cows with subclinical mastitis revealed a highly significant increase in sodium (P < 0.001). Comparison of healthy cow's milk with that of clinically mastitic milk showed a highly significant decrease in levels of calcium, magnesium (P < 0.001) and potassium (P < 0.01). However, sodium increased highly significantly (P < 0.001). Comparison of macro-minerals in milk from cows with subclinical and clinical mastitis revealed a significant decrease in potassium contents (P < 0.05) compared with that of healthy cows. Potassium levels were found to decrease significantly (P < 0.05) in subclinically infected cow's blood serum. However, calcium and phosphorus showed a significant decrease (P < 0.01) in blood serum samples from the clinically infected cows.     

Antipenicillinase in the serum of dairy cows

Serum from dairy cows was tested for inhibitory effect on penicillinase from a penicillin-resistant Staphylococcus aureus strain of mastitic origin. Among cows with subclinical mastitis caused by penicillin-resistant S. aureus there was a significantly higher frequency of individuals with penicillinajse-inhibiting serum than among healthy cows. Among the subclinical cases, a foregoing penicillin treatment of clinical mastitis appeared to increase the serum antipenicillinase activity.     

Effect of intramammary infusion of rbGM-CSF on SCC and expression of polymorphonuclear neutrophil adhesion molecules in subclinical mastitis cows

The effect of rbGM-CSF intramammary infusion on the subclinical mastitis was evaluated by the somatic cell count (SCC) and expression of adhesion molecules (CD62L and CD11b) on the surface of neutrophils (PMN) in blood and milk. Fifteen cows diagnosed to have subclinical mastitis were used in this study. Seven cows showed a decrease in the SCC (decreased group), whereas 8 cows showed an increase in the SCC (increased group) 7 days after infusion of rbGM-CSF compared to pre infusion level. The percentage of CD62+ cells tended to be lower and CD11b+cells tended to be higher at 6 h on blood PMN in the decreased group of cows. Increased group of cows showed opposite tendencies. The mean fluorescent intensity of these adhesion molecules expressed on PMN in blood and milk was similar in both groups. These results suggested some association between expression of adhesion molecules and changes in SCC by rbGM-CSF. Responsiveness of PMN adhesion molecules to rbGM-CSF might determine the changes in SCC of the subclinical mastitic cows after infusion of rbGM-CSF.