Effect of pre- and postpartum supplementation with lipid-encapsulated conjugated linoleic acid on reproductive performance and the growth hormone-insulin-like growth factor-I axis in multiparous high-producing dairy cows.

The objective of the study was to evaluate the effect of prepartum and postpartum (PP) supplementation with 2 isomers of conjugated linoleic acid (CLA) on reproductive parameters and some related metabolic factors in dairy cows. High-producing, multiparous Holstein Friesian cows (n = 60) were allotted to 3 treatment groups: the CLA1 group (n = 20) was supplemented with 70 g of lipid-encapsulated CLA providing 7 g each of cis-9,trans-11 and trans-10,cis-12 CLA from d 21 (d 21) before expected calving until d 7 after artificial insemination (AI), that is, until 77 to 91 d PP; the CLA2 group (n = 20) was supplemented with the same amount of CLA beginning at calving until d 7 after AI; and the control group (n = 20) received an isocaloric, isonitrogenous, and isolipidic diet. Blood samples were taken weekly to measure glucose, insulin, insulin-like growth factor-I (IGF-I), and leptin. Liver biopsy was performed in 10 cows per group for growth hormone receptor 1A and IGF-I mRNA analyses. At d 49 to 63 PP, ovulation was synchronized with the Pre-Synch protocol followed by fixed-time AI. Milk progesterone was monitored from calving until d 35 post-AI. Cows returning to estrus following AI were inseminated. Supplementation with CLA before calving improved the recovery of plasma leptin levels in the early PP period (from the day of calving until wk 3 PP; treatment effect). Later PP (wk 5), plasma IGF-I, and leptin remained significantly higher in both CLA1 and CLA2 groups compared with control, although hepatocellular IGF-I mRNA was not different among groups. Plasma IGF-I levels remained higher in both CLA-treated groups on the day of AI. Growth hormone receptor 1A mRNA levels in hepatic tissue decreased in all groups, reaching a nadir in the first week PP. Days to first PP ovulation did not differ between groups; however, both supplemented groups conceived earlier compared with control (d 97 ± 19, d 97 ± 23, and d 113 ± 30 for CLA1, CLA2, and control, respectively). Plasma progesterone concentration was higher in both supplemented groups on d 2 to 5 following the synchronized ovulation than in controls. We concluded that CLA supplementation around calving alters PP metabolic signals as reflected by higher plasma leptin and IGF-I levels. Conjugated linoleic acid stimulated early luteal function and reduced the PP interval to conception.

Subsequent effect of subacute T-2 toxicosis on spermatozoa, seminal plasma and testosterone production in rabbits.

Pannon White (n=12) male rabbits (weight: 4050 to 4500 g, age: 9 months) received 2 ml of a suspension containing purified T-2 toxin by gavage for 3 days. The daily toxin intake was 4 mg/animal (0.78 to 0.99 mg/kg body weight (BW)). Control animals (n=12) received toxin-free suspension for 3 days. Since a feed-refusal effect was observed on the second day after T-2 administration, a group of bucks (n=10) were kept as controls (no toxin treatment) but on a restricted feeding schedule, that is, the same amount of feed was provided to them as was consumed by the exposed animals. On day 51 of the experiment (i.e. 48 days after the 3-day toxin treatment), semen was collected, and pH, concentration, motility and morphology of the spermatozoa, as well as concentration of citric acid, zinc and fructose in the seminal plasma, were measured. After gonadotropin-releasing hormone (GnRH) analogue treatment, the testosterone level was examined. One day of T-2 toxin treatment dramatically decreased voluntary feed intake (by 27% compared to control, P<0.05) and remained lower (P<0.05) during the first 2 weeks after the withdrawal of the toxin. BW of the contaminated rabbits decreased by 88% on days 17 and 29 compared to controls (P<0.05). No effect of toxin treatment was detected on pH and quantity of the semen or concentration of spermatozoa. The ratio of spermatozoa showing progressive forward motility decreased from 65% to 53% in the semen samples of toxin-treated animals compared to controls (P>0.05). The ratio of spermatozoa with abnormal morphology increased (P<0.05) in the ejaculates collected from the toxin-treated animals. T-2 toxin applied in high doses decreased the concentration of citric acid in seminal plasma (P<0.05). No effect of T-2 toxin on the concentrations of the other seminal plasma parameters (fructose and zinc) was observed. T-2 toxin decreased the basic testosterone level by 45% compared to control (P<0.01) and resulted in lower (P<0.05) GnRH-induced testosterone concentration. Feed restriction, that is, less nutrient intake, resulted in more morphologically abnormal spermatozoa in the semen, but it did not cause significant loss in BW, motility of the spermatozoa, composition of the seminal plasma or testosterone concentration--its effect needs further examination.

AluI polymorphism of the bovine growth hormone (GH) gene, resumption of ovarian cyclicity, milk production and loss of body condition at the onset of lactation in dairy cows.

Relationships among GH genotype (AluI polymorphism), parity, metritis and interval from calving to first ovulation, milk production and body condition score (BCS) loss were determined in dairy cows (n=307) on four large-scale farms in Hungary. Cows with systemic signs of puerperal metritis or mastitis were excluded. Time of the first postpartum (PP) ovulation was obtained from milk progesterone profiles. Based on GH genotype determination, groups of leucine homozygous cows (n=246) and valine allele carriers (n=61) were formed. All animals became cyclic during the study period. The average interval to first ovulation was 27.6+/-0.69-d PP (mean+/-S.D.). Genotype had no effect on the commencement of ovarian cyclicity. First ovulation occurred sooner after calving in pluriparous than in primiparous cows. The greater BCS loss cows had during the first 30-d PP, the longer they took to resume cyclic ovarian function. The interval from calving to first ovulation was substantially affected by farm, but not by mild cases of puerperal metritis. Genotype was not related to cumulative 30-d milk yield or BCS loss after calving. Primiparous cows had lower milk yield than pluriparous ones. Cows with metritis lost more body condition than healthy individuals in the first month postpartum. We concluded that, under field conditions, AluI polymorphism of the bovine GH gene had no effect on the interval from calving to first ovulation and could not be directly related to differences in milk yield and to the extent of BCS loss during the first month after calving in Holstein-Friesian cows.

Bacterial complications of postpartum uterine involution in cattle.

The bacterial contamination of the postpartum uterus is a frequent finding which by itself does not disturb the anatomical and histological restoration of tubular genital tract. The improper balance between uterine infection and the intrauterine antimicrobial self-defence mechanisms, however, often results in complications, such as puerperal metritis, clinical endometritis, pyometra and subclinical endometritis. After reviewing the bacteriology of uterine involution, and the predisposing factors for its bacterial complications, this paper defines the different clinical forms, and summarizes their pathology, furthermore, the recent progress in diagnostic considerations and principles of current treatments for these diseases of bovine genitals.

Pregnancy stage and number of fetuses may influence maternal plasma leptin in ewes.

Maternal plasma leptin is elevated in ewes during pregnancy. The authors studied whether there was any relation between maternal plasma leptin and insulin concentrations, the number of fetuses and the circulating and faecal levels of gestagens. At the end of the breeding season in January the ovarian activity of Prolific Merino ewes was induced/synchronised with gestagen + eCG treatment. Ewes were inseminated artificially (AI) by laparoscopy. Blood and faecal samples were collected before AI (day 0) and again 41, 81 and 101 days later. The plasma levels of leptin (pL), insulin and progesterone (pP4), and the faecal P4 metabolite (P4-met) content were determined. The day 0 level of pL was significantly higher in pregnant (n = 24) than in non-pregnant ewes (n = 32). By day 41 the pL of pregnant animals had doubled, it showed a further moderate increase on day 81, and decreased slightly thereafter. During pregnancy pP4 and faecal P4-met rose continuously and were positively correlated at all stages. The mean levels of pL and pP4 and the faecal content of P4-met were lower in ewes bearing single (n = 12) than in those with 2 (n = 6) or 3-5 fetuses (n = 6). Analysis of variance demonstrated significant differences according to the number of fetuses in the pL and pP4, but not in P4-met (p = 0.042, 0.044, and 0.051, respectively). Leptin showed positive correlation with insulin before the AI but not during pregnancy. On days 41 and 81 pL showed a slight positive correlation with P4 and P4-met, which decreased slightly by day 101. This study shows that although leptinaemia is affected by the number of fetuses and the level of P4, pregnancy stage is a more important regulator than these additional factors.

Endocrine characteristics of late pregnant hyperketonaemic ewes and their reproductive performance following the induction of ovarian cyclicity out of the breeding season.

Ketosis was diagnosed in a flock of Merino ewes that conceived from synchronised oestrus in the early autumn period. On day 140 of pregnancy the ewes were sampled for determination of betaOH-butyrate (BHB), AST, glucose, non-esterified fatty acids (NEFA), total cholesterol (TCH), insulin, T4, T3, cortisol, IGF-1 and leptin. The results were evaluated according to the number of fetuses born some days later and the presence of hyperketonaemia (BHB: > or = 1.60 mmol/l). In May, about 3 months after lambing, cyclic ovarian function was induced (Cronolone + eCG), and the ewes were inseminated artificially (AI) 48 h after the removal of gestagen-containing sponge. At the time of AI and 10 days later blood samples were collected again to check the plasma levels of the same constituents as previously (in samples taken at AI), and to monitor the ovarian response by assaying progesterone (in both samples). On day 140 of gestation significantly lower BHB levels were detected in dams with single (n = 41) than in those with twin (n = 57) pregnancies. Hyperketonaemia was found only in ewes bearing twins (n = 27). These animals had higher NEFA and cortisol, and lower TCH, insulin, IGF-1, leptin and T3 levels than their normoketonaemic twin-bearing flock-mates, and those with single pregnancy. The blood glucose concentrations varied within a wide range, and the means of groups did not exhibit any significant differences. The formerly hyperketonaemic individuals were characterised by lower leptin level 3 months after lambing, and they showed a poorer response to the cycle-induction procedure than the others. The non-responders had lower IGF-1 and leptin levels than those ovulated after this treatment. It was concluded that the subclinical form of ovine ketosis is characterised by complex endocrine alterations, reflecting an obvious form of negative energy balance. If attempts to induce cyclic ovarian function outside the breeding season are made soon after lambing, the ovarian response and fertility of these ewes may also be depressed.

Effects of clinical mastitis on ovarian function in post-partum dairy cows.

Mastitis-induced ovarian abnormalities were studied in a field trial. At 1-3 day after calving, > or = 2 parity cows not affected with chronic recurrent mastitis and yielding < 400,000/ml somatic cell count (SCC) individual milk in the previous lactation, were enrolled in the study. Thereafter milk samples were collected three times weekly for 95-100 day for progesterone (P4) assay. Individual P4 profiles were used to monitor ovarian cyclicity. When mastitis was diagnosed in the first 80 day post-partum (pp), clinical signs were recorded and scored, and aseptic milk samples were taken to identify the mastitis pathogens. Depending on the isolated pathogens the cows were blocked into one of the three sub-groups affected by either Gram-positive (GP), or Gram-negative (GN) bacteria, or of those with no detected pathogens (NDP). Cows suffering from any type of mastitis between days 15 and 28 (n = 27) showed a delay in the onset of ovarian cyclicity, and estrus was postponed compared to cows affected during the first 14 day pp (n = 59) and controls (n = 175) (38.6 +/- 2.3 vs 33.4 +/- 2.1 and 32.0 +/- 1.0 day, respectively, for onset of ovarian cyclicity and 90.7 +/- 2.5 vs 80.2 +/- 2.8 and 83.9 +/- 2.1 day, respectively, for estrus; both p < 0.05). The percentage of cows ovulating by day 28 was lower in those affected by mastitis between days 14 and 28 compared to cows between days 1 and 14 and controls (22.2% vs 47.5 and 50.3%, respectively; p < 0.05). A significantly higher rate of premature luteolysis was observed in GN + NDP compared to GP mastitis and healthy cows (46.7% vs 8.3 and 2.0%, respectively; p < 0.001). If the mastitis outbreak occurred during the follicular phase, the duration of this cycle segment was lengthened in GN + NDP mastitis compared to GP mastitis and healthy cows (10.8 +/- 0.9 vs 7.9 +/- 0.1 and 7.2 +/- 0.1, respectively; p < 0.001). The results indicate that mastitis can affect the resumption of ovarian activity in pp dairy cows. Mastitis may also impair reproduction also in cyclic cows: this effect can be the consequence of premature luteolysis or a prolonged follicular phase.

Feeding-unrelated factors influencing the plasma leptin level in ruminants.

The triglyceride content of lipid depots associated with the current feeding level is the primary determinant of leptin gene expression and the circulating leptin level. In laboratory rodents and primates the plasma leptin is influenced also by the age, gender and physiological status (puberty, pregnancy, lactation, postpartum period), and by the health condition such as sepsis due to Gram-negative (GN) bacteria. Some pathologic conditions with intensive cytokine release evoke an increase in plasma leptin, which is thought to depress the subsequent feed intake. However, the effect of these secondary factors may be species-dependent, with still unknown clinical relevance in ruminants. In our ovine and bovine models plasma leptin increased after castration and dexamethasone treatment, decreased after experimental administration of synthetic androgens in castrated rams, but remained unchanged throughout the ovarian cycle and after ovariectomy. The circulating leptin level increased temporarily during synthetic progestin (fluorogestone) treatment in ewes, but similar changes were not seen in progesterone-supplemented ewes and norgestomet-treated cows. In a second trial on dairy cows we wanted to study whether elevated plasma leptin levels are induced by experimental endotoxin mastitis, or by natural outbreak of GN mastitis and puerperal metritis. Experimental endotoxin mastitis resulted in some-hour elevation in cortisol and insulin, with a simultaneous decrease in IGF-I and thyroid hormones. In the first 14 days of lactation GN mastitis induced the same endocrine alterations as the experimental endotoxin challenge, but in natural cases these changes varied within a wider range, and were more protracted and robust. Cows with puerperal metritis had more obvious catabolic changes in the early weeks of lactation, than their healthy counterparts. However, both mastitis and puerperal metritis failed to increase the circulating leptin level, showing that in cows the plasma leptin is not responsible for the anorexia associated with these inflammatory diseases.

Factors affecting plasma progesterone concentration and the retrospective determination of time of ovulation in cyclic mares.

Factors influencing plasma progesterone concentration were investigated in seven mares. Two-phase logistic curves were fitted (r=0.98) to plasma progesterone concentrations of blood samples collected once daily. In addition to the effect of time (P<0.001), there were differences (P<0.01) among mares in the peak height of the progesterone plateau and in the (area under the curve) AUC. Plasma progesterone concentrations were higher (P<0.001) after a multiple versus single ovulation. There was an effect of season (P<0.001), but no significant effect of luteal morphology. The retrospective determination of time of ovulation was carried out using a linear model on the seven mares and 25 additional mares. Linear regression on the measured values or on the ratio to the average concentration from D5 to D10, was calculated with the day of cycle between D0 and D4. The ovulation date was then calculated using both of these equations, whether blood sampling was performed twice or thrice weekly on 25 postpartum mares. The accuracy to predict day of ovulation (+/- 1 day) ranged from 88 to 97%. In conclusion, the retrospective estimation of time of ovulation in mares was possible, although the technique had some limitations.

Energy imbalance related predisposition to mastitis in group-fed high-producing postpartum dairy cows.

The energy imbalance related predisposition to mastitis was studied in group-fed postpartum dairy cows (n = 333) kept in 4 large-scale units and producing milk of low somatic cell count (SCC). Blood samples were taken on Days 1-3 after calving for assaying some metabolites and hormones related to the negative energy balance (NEB). If mastitis was diagnosed later, aseptic milk samples were taken to identify the pathogens. Considering pathogen types [contagious pathogens: Staphylococcus (S.) aureus, Gram-positive (GP) environmental pathogens, and Gram-negative (GN) environmental pathogens + mastitis with no detectable pathogens (NDP)] separately, stepwise logistic regression was used to analyse the relation between the potential prognostic value of hormones and metabolites and mastitis outbreak. Only the elevated (> or = 1.00 mmol/l) serum beta-hydroxybutyrate (BHB) levels predisposed the cows to mastitis in the subsequent 4 weeks. This prognostic value of BHB was significant only in GN + NDP mastitis and in cases caused by GP environmental pathogens, but not in S. aureus mastitis (odds ratio: 5.333, 3.600 and 1.333, respectively).

Periparturient endocrine and metabolic changes in healthy cows and in cows affected by mastitis.

Transition from pregnancy to lactation in dairy cows involves considerable metabolic adaptation. Additional stress is incurred during infections such as periparturient mastitis. Multiparous Holstein-Friesian cows kept under normal production conditions (n = 15) were used to evaluate changes in circulating metabolite and hormone concentrations from 5 days before to 5 days after calving. Insulin-like growth factor binding protein (IGFBP) profiles were also monitored. Marked time-related changes were observed for plasma thyroid hormone, IGF, cortisol, insulin, beta-hydroxybutyrate (BHB) and non-esterified fatty acid (NEFA) concentrations but not for plasma leptin. A decrease in IGF-II concentration and maximal intensity of the putative IGFBP-1 band occurred at parturition. When compared with the five healthy cows,low IGF-II levels were prolonged to day 2 post-partum in five cows with Escherichia coli-associated mastitis. However, marked decreases in IGFBP-2 band intensity were evident only in two of the four cases examined. Individual total ligand (IGF-I + IGF-II) concentration and IGFBP pattern prepartum were largely regained 5 days post-partum in all cows. Hormone and metabolite concentrations in the two cows with Staphylococcus aureus-associated mastitis were very similar to those in the five healthy cows. Plasma thyroxine (T4) was lower 2 days prepartum in the cows, which later developed Gram-negative mastitis. Multiregression analysis showed that variance in T4 concentration was significantly and independently associated with triiodothyronine (T3) and IGF-I positively and with cortisol negatively (R2 = 0.648). This study confirms the close inter-relationship between the thyroid hormone and IGF axes in cattle and indicates possible effects of Gram-negative mastitis infection on IGF-II metabolism.