G. Stradaioli

Glycoconjugates in small antral ovarian follicles of the river buffalo (Bubalus bubalis L.)

Types and distribution patterns of glycoconjugates in antral ovarian follicles were investigated in the buffalo, using periodic-acid Schiff (PAS), high iron diamine (HID), low ion diamine (LID) and lectin histochemical staining methods. HID and LID staining procedures were preceded in some cases by digestion with testicular hyaluronidase, Streptomyces hyaluronidase, chondroitinase ABC and heparitinase (heparinase III). Lectin staining was performed with the use of 12 horseradish peroxidase (HRP) lectin conjugates. Some lectin staining procedures were preceded by neuraminidase digestion and saponification. Large amounts of isomeric chondroitin sulphates and a minor quantity of heparan sulphate and hyaluronic acid and/or chondroitin were found in follicular fluid. Lectin staining of buffalo follicular fluid revealed glycoconjugates with different glucidic determinants such as beta-N-acetylgalactosamine, beta-galactose-(1-3)-N-acetylgalactosamine, beta-galactose-(1-4)-N-acetylglucosamine, N-acetylglucosamine, alpha-fucose and alpha-glucose/alpha-mannose, and sialic acid residues. Glycosaminoglycans were absent in the zona pellucida of oocytes in small antral follicles. Acidic glycoconjugates in the zona pellucida were caused by sulphated groups and sialic acid residues. Our data show few internal glucidic residues, such as N-acetylglucosamine in the buffalo zona pellucida but many subterminal beta-N-acetylgalactosamine, alpha- and beta-galactose determinants masked by sialic acids. These findings demonstrate that buffalo follicular fluid has a very heterogeneous composition that is similar to that found in small and large bovine follicles. No differences in composition of the follicular fluid were observed in the follicles examined.

Phospholipid hydroperoxide glutathione peroxidase in bull spermatozoa provides a unique marker in the quest for semen quality analysis.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoperoxidase accounting for most of the selenium content in mammalian testis, which has been found to be linked to fertility in humans. In this study, we addressed the issue whether PHGPx content in spermatozoa could be a predictive index of fertilization capacity for sire selection in bulls. Measurement of PHGPx in spermatozoa of 92 yearling bulls of three different Italian breeds (Chianina, Romagnola, and Marchigiana) revealed the presence of two quite well separated populations. A PHGPx activity of 130 mU/mg separated the high-PHGPx group (H-PHGPx, n=73) from the low-PHGPx group (L-PHGPx, n=19). Forward motility was markedly higher in the H-PHGPx group, which also contained a lower percentage of detached heads, abnormal midpiece, and proximal droplets. On the other hand, differently from the human studies, no correlation was observed between PHGPx activity and number of spermatozoa in the ejaculate. Apart from sperm count, which typically differed among breeds, and number of detached heads in the L-PHGPx group, which correlated with higher sperm count, no other significant difference in seminal parameters among breeds was apparent. The assay for sperm PHGPx activity therefore emerges as a unique tool to evaluate semen quality for sire selection.

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.

Factors affecting milk cortisol in mid lactating dairy cows

BackgroundWhether the measurement of cortisol in dairy cows can be used as a biomarker of adverse environmental or pathophysiological conditions is still under of scientific debate. In these situations, several systems mainly the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, and the immune system are recruited to reestablish homeostasis. A first aim of the present study was to compare milk and blood cortisol concentrations and to consider its variability in milk in relation to farm, milk yield and days in milk. A second study investigates the effects of breed, class of somatic cell count (SCC) and farm on milk cortisol levels in a larger number of cows and farms, with the aim to validate the results obtained in the pilot study.MethodsFor study 1, 135 cows were sampled from 2 Italian Simmental and 2 Italian Holstein commercial farms, whilst in the second study, 542 cows were sampled from 6 commercial farms of Italian Simmental and 499 cows from 4 commercial farms of Italian Holstein.ResultsIn study 1, the values of cortisol content in milk were significantly higher in Holstein than Simmental cows. Significant differences between farms were observed for milk and plasma cortisol concentrations. Cortisol content in milk was not correlated to plasma content in study 1 and the mean milk to plasma cortisol ratio was about 1:30. In study 2, for Holstein cows, significantly higher values of milk cortisol in comparison to Simmental cows was reported. A significant effect of class of SCC was observed, cows belonging to class 3 (SCC higher than 400.000/ml) showed the highest mean values of milk cortisol. The farm effect was significant also in the study 2, confirming the results obtained in the first study.ConclusionsMilk can be considered a preferential site of sampling in dairy cows to point out short term stimulation of the hypothalamic-pituitary-adrenal axis. Further studies are needed to investigate the physiological basis of the relationship between milk cortisol content and breed, milk yield and SCC to ascertain the relevance of milk cortisol to monitor the healthy status of mammary gland.

Reproductive performance of dairy cows with luteal or follicular ovarian cysts after treatment with buserelin

In dairy farm management economic losses resulting from cystic ovarian degeneration are well known. In spite of this, neither the definition nor the aetiopathology of ovarian cysts are clear and agreed upon. Also the usual classification in luteal and follicular cysts, requiring ultrasound examination together with assessment of P4 to be accurate, is not very helpful in field conditions. Consequently a single treatment is often provided for both types of cysts, and since the 1970s treatments with GnRH and its analogues have been considered very useful. Nevertheless differences in recovery rates after GnRH treatment in animals with either luteal or follicular cysts are reported. Thus, the aim of this study was to evaluate recovery rate, recovery time and conception rate after treatment with buserelin (GnRH-analogue) in cows with ovarian luteal or follicular cysts. In a 5 years period, 150 cows with cysts out of a total of 990 animals, were detected and treated intravenously between 45 and 60 days PP with 20μg buserelin. No statistically significant differences were found in recovery rates and in conception rates between the two types of cysts. Comparison of recovery times showed significantly shorter recovery for cows with luteal cysts. The results emphasise the usefulness of GnRH to treat ovarian cysts regardless of their type, in relation to both recovery and conception rates. Intervals from treatment to resumption of ovarian activity were affected by the characteristics of ovarian cysts, with a faster recovery for the luteal type.

Dietary grape poliphenols modulate oxidative stress in ageing rabbits

Riassunto I polifenoli dell’uva regolano lo stress ossidativo nei conigli durante l’invecchiamento. Lo studio ha avuto come obiettivo di verificare gli effetti di un’integrazione alimentare con polifenoli dell’uva sul controllo dello stress ossidativo in conigli durante l’invecchiamento. A tal fine sono stati predisposti 4 gruppi sperimentali che ricevevano diverse concentrazioni giornaliere di polifenoli (CTR: 0 mg/kg di mangime; P1: 0,03 mg/kg; P2: 0,15 mg/kg; P3: 0,30 mg/kg). I prelievi di sangue sono stati effettuati a 1 (T1), 6 (T6) e 10 (T10) mesi dall’inizio della somministrazione dei polifenoli, da settembre 2003 a luglio 2004. Come indicatori di stress sono stati utilizzati il glutatione totale (GSx), il ridotto (GSH) e l’ossidato (GSSG). I livelli di glutatione totale e ridotto sono diminuiti in T6 per poi aumentare nuovamente in T10. I livelli di glutatione ossidato non hanno subito variazioni significative nel tempo. I valori di glutatione totale, ridotto e ossidato hanno mantenuto livelli significativamente più bassi nel gruppo P3 rispetto agli altri gruppi sperimentali. Questi risultati indicano che i polifenoli dell’uva hanno, anche nei conigli, un’azione di regolazione dell’omeostasi ossido-riduttiva.

Reducing treatments in cattle superovulation protocols by combining a pituitary extract with a 5% hyaluronan solution: Is it able to diminish activation of the hypothalamic pituitary adrenal axis compared to the traditional protocol?

Traditional superovulation protocols that include multiple gonadotropin treatments are time-consuming and labor intensive. These protocols require multiple handling and restraining of embryo donors. This will likely increase the risks of injuries in both animals and humans and induce stress that may lead to a reduced superovulatory response. These are more evident when working with cattle that are rarely handled or raised on extensive grazing. The objectives of this experiment were to compare the efficacy of a split-injection protocol of porcine pituitary-derived porcine FSH (pFSH) preparation (slow release [SR] group) to the traditional 4-day treatment with pFSH administered twice daily (C group) and to determine the concentrations of cortisol in the hair as a marker of activation of the hypothalamic-pituitary-adrenal (HPA) axis during the two superovulatory treatments. Thirty-two heifers were stimulated twice in a 2 × 2 crossover design and compared for ovarian response and numbers and characteristics of recovered ova-embryo among treatments. No differences between SR and C groups were found in terms of percentage of responsive animals (100% vs. 93.8%) and ovulation rate (83.7 ± 1.1 vs. 79.5 ± 1.0%). A positive correlation was found between the number of follicles responsive to pFSH (2-8 mm) at the beginning of treatments and the superovulatory response, and no differences were found in these follicular populations between the two treatment groups. The numbers of CLs, ova-embryos, fertilized ova, transferable and freezable embryos recovered per cow were found to be significantly higher in SR compared with C group (14.0 ± 1.6 vs. 10.6 ± 1.0, 12.1 ± 1.6 vs. 7.6 ± 1.0, 11.1 ± 1.1 vs. 7.3 ± 1.0, 9.6 ± 1.4 vs. 6.6 ± 1.0, and 9.4 ± 1.4 vs. 6.0 ± 1.0 for SR and C group, respectively). The SR group produced also a significantly greater number of excellent- and/or good-quality embryos compared with the C group. The concentrations of cortisol in the hair at Days 14 and 21 were significantly greater in the C compared with the SR group (P < 0.05). In conclusion, these results indicate that the dilution of gonadotropin in a 0.5% hyaluronan solution, reducing the administration frequency, improves the quantitative and qualitative superovulatory response of Marchigiana heifers [corrected]. Further studies using other breeds of cattle are needed to verify the results herein obtained and to confirm the lower activation of the hypothalamic-pituitary-adrenal axis caused in the donor by the split-injection protocol.

Ultrasonographic features of the mule embryo, fetus and fetal-placental unit

The aim of this study was to establish baseline ultrasound data concerning the mule conceptus during gestation. Ten multiparous Trotter mares were artificially inseminated with chilled semen from an Amiatino jack donkey. Daily transrectal ultrasonography was carried out from the day of ovulation until Day 50 of gestation to determine the following: first detection of the embryonic vesicle (EV), mobility phase, EV diameter, day of EV fixation, changes in EV shape, date of yolk sac regression and embryo crown-rump length. Monthly ultrasonic assessments from Day 50 of gestation to term were carried out. These assessments included an evaluation of fetal well-being and the growth of the mule conceptus, which were monitored using the following variables: cardiac activity, fetal activity and presentation, fetal fluid echogenicity, combined thickness of the utero-placenta unit and fetal orbital and aortic diameter. Mule EV first detection was observed earlier (37% at Day 8) than that observed in the equine pregnancy. EV diameter at first detection was 4.6 ± 1.1 mm. At Day 10, 75% of EVs were detected. EV fixation occurred on Day 17.1 ± 1.1, with a mean EV diameter of 2.5 ± 0.2 cm. EV growth rate was 4.04 mm/day from Days 11 to 16, 0.4 mm/day from Days 16 to 28 and 1.78 mm/day from Days 28 to 45 of pregnancy. The embryo proper was first detected on Day 19.9 ± 1.9 (average length 2.4 ± 1.4 mm), and the embryonic heartbeat was first detected on Day 24 ± 2.4. The fetal carotid pulse was observed at six months of gestation and provided a good means by which to estimate fetal cardiac activity in advanced gestation. The fetal heart rate was recorded from Month 2 of gestation to term. The mean ± SD of the combined uteroplacental thickness was assessed at the cervical-placental junction and at the ventral abdomen in mares between Months 2 and 5 until term, respectively. An abnormal fetal-placental unit and fetal inactivity was observed in association with abortion. Mule-conceptus biometric measurements correlated significantly with the gestational age, and these data were used to predict an unusually large mule fetus, which might result in dystocia. In conclusion, we can assume that early diagnosis of pregnancy failure and assessment of fetal biophysical profile and growth charts could improve the chances of gestation completion in mule-pregnant mares. The early detection of mares at risk for an abnormal pregnancy or delivery may increase the success of prompt treatments, therefore preventing costly emergency procedures and allowing proper obstetrical and neonatal assistance.

Hair cortisol and testosterone concentrations and semen production of Bos taurus bulls

Abstract The experiment was aimed to evaluate the usefulness of hair cortisol concentrations (HCC) in revealing the activation of the hypothalamic-pituitary-adrenal (HPA) axis due to the passage of young bulls from genetic rearing stations to artificial insemination (AI) centre and the influence of this passage on hair testosterone concentrations (HTC). Hair samples on 33 yearling bulls were collected at the time of arrival (ST1) to the AI centre and 3 more hair samples (ST2, ST3, ST4) were collected at monthly intervals. After the end of quarantine each animal was submitted to the standard procedures for collection and production of semen. Overall HCC were significantly affected by period of collection (p < .01). In particular, HCC was higher in samples obtained after finishing the quarantine period (ST2) compared with those obtained on ST1. After ST2, the HCC decreased reaching at ST4 a value similar to that recorded at ST1. An effect of sampling time on HTC was not found (p > .05). The effect of breed was observed only for HTC that was higher in PRI than BS (p < .01). The HCC group have not influenced the semen variables (p > .05). On the contrary, the effect of breed was evident. The results of this study replicated the effects of a major environmental stressor on mean cortisol concentrations assessed in hair, and add to the growing body of evidence that HCC is an effective and simply collected marker for long-term activity of the HPA system in response to persistent environmental challenge.

Apoptosis and Expression of Related Proteins in Mammary Gland of Heifers during Early Lactation

Lactation is biologically significant and economically important and it has particular nutritional implications in the case of dairy cows. Programmed cell death is a physiological event during mammary gland involution and also plays an important role in the regulation of milk production and persistence of lactation (Capuco and Akers, 1999), although it is numerically less relevant (4.8% vs 2.4%; Wilde, personal communication; Quarrie et al., 1995). Proteins belonging to the bcl-2 family, namely bax and bcl-2, are involved in the regulation of cell apoptosis (Reed, 1997) which also occurs in the mammary glands of lactating mice (Heermeier et al., 1996) and sheep (Colitti et al., 1999); bax acts as a pro-apoptotic and bcl-2 as an anti-apoptotic agent. The aim of the current research was to investigate the morphological organization and the extent of cell apoptosis and proliferation of mammary cells in heifers during early lactation. The possible relationship between mRNA expression of bax and bcl-2 and cell fate was also studied.