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Intrauterine laparoscopic insemination is diffuse in sheep breeding, yet requires veterinary expertise and expensive equipment. Our aim was to evaluate the time required, reproductive outcome and post-operative complications of mini invasive laparotomic intrauterine insemination, as an alternative approach to laparoscopy. A total of 115 Sopravvissana breed ewes were enrolled, including 75 adults and 40 lamb ewes, after oestrus synchronisation. Ewes were sedated and restrained in dorsal recumbence. Uterine horns were exposed through an abdominal incision of 4–7 cm, cranially to the udder. Thawed semen, containing 100 × 106 progressive motile (70%) and morphologically normal (80%) sperm cells, was inoculated into uterine lumen through an intravenous catheter. The abdominal incision was then routinely closed. The time interval for: (a) laparotomy and uterine exteriorisation, (b) intrauterine insemination, (c) abdominal wall closure and (d) total procedure was recorded. Pregnancy, lambing, twinning rates and sex ratio were submitted to chi-square test, comparing lamb and adult ewes. Time intervals and the duration of laparotomic procedure were compared in lamb and adults through Mann–Whitney U test for independent samples. Median ± standard error (SE) of total time for laparotomic intrauterine insemination was 5.53 ± 0.07 and 4.99 ± 0.11 min, for adult and lamb ewes (p

The objective of the present study was to evaluate the effect of 24 h’ fasting prior to semen collection by electroejaculation on behavioral responses, volume of rectal fecal content, bladder size, penis protrusion, erection, ejaculation stimuli, and ejaculate parameters in young Simmental bulls. Twenty-two Simmental beef bulls with an age of 13.2 ± 1.2 mo (mean ± SD) were used in a prospective randomized blinded controlled cross-over design with two pens fasted for 24 h (n = 9; FAS group), and the other three pens were non-fasted (n = 13; CON group). The bulls were maintained under confined conditions without access to pasture. One week later, the pen treatments were inverted, and semen was collected again under the same conditions and by the same team. The behavioral responses, volume of fecal rectal content, bladder size, as well as the number of stimuli required to obtain penis protrusion, erection, and ejaculation to electroejaculation were measured. The following ejaculate parameters were measured: volume, concentration, spermatozoa motility, and morphology. The behavioral response of the bulls to electroejaculation was not different between the CON group and the FAS group (3.2 ± 0.5 and 3.0 ± 0.7, respectively; P = 0.36). Bladder size was significantly reduced in the FAS group compared with the CON group (2.3 ± 0.8 vs. 2.8 ± 0.9, respectively; P = 0.02). The volume of feces in the rectum was not different between the two groups (CON was 2.3 ± 1.7 and FAS was 3.0 ± 1.8; P = 0.23). Compared with the CON group, the FAS group showed a higher proportion of penis protrusion (100% versus 81.8%, P = 0.10), erection (100% versus 81.8%; P = 0.10), and ejaculation (100% versus 90.9%; P = 0.49). The combined efficiency of penis protrusion, erection, and ejaculation (CE-PPEE) in the FAS group was superior to that of the CON group (P = 0.001). The number of stimuli necessary for penis protrusion, erection, and ejaculation for the CON group were 13.5 ± 3.7, 14.9 ± 3.7, and 20.8 ± 5.8, and they were 15.0 ± 4.2, 16.6 ± 4.2, and 20.2 ± 8.1 for the FAS group. The number of stimuli for penis protrusion (P = 0.09), erection (P = 0.08), and ejaculation (P = 0.77) were no different between the two groups. Ejaculate volume was 4.0 ± 2.6 ml and 4.1 ± 2.3 ml for the CON and FAS groups, respectively (P = 0.90). The motility was 1.4 ± 0.7 and 1.4 ± 0.8 for the CON and FAS groups, respectively (P = 0.72). The concentration of spermatozoa was 336.2 ± 273.1 million and 421.1 ± 300.6 million for the CON and FAS groups, respectively (P = 0.31). The percentage of normal spermatozoa was 50.9 ± 18.8 and 45.6 ± 14.3 for the CON and FAS groups, respectively (P = 0.16). It was concluded that fasting for 24 h prior to semen collection by electroejaculation reduced the bladder size and increased the proportion of bulls with penis protrusion, erection, and ejaculation without any difference detected in behavioral responses, volume of rectal fecal content, and ejaculate parameters.

The aim of the study was to evaluate the net return of the implementation of a remote calving monitoring system for obstetrical and neonatal assistance on the herd economy in a dairy farm model. A total of 680 parturitions over a 7-yr period were evaluated. Age at first calving was restricted from 23 to 27 mo for primiparous cows to be included. Among groups of cows that were ready to calve in a 15-d interval, primiparous and multiparous were randomly assigned to the experimental group and monitored through a calving alarm system, whereas the others accounted for controls. Final parturition groups were as follows: control primiparous (CPP, n = 218), control multiparous (CM, n = 345), monitored primiparous (MPP, n = 56), and monitored multiparous (MM, n = 61). Monitored groups received prompt calving assistance and first neonatal care, whereas the presence of farm personnel was discontinuous for controls. A biological model was built considering significant differences in calf loss, early culling, milk production, and days open between groups. Then, a partial budget model was used to estimate costs and net return on a simulated herd of 100 lactating cows. Incidence of calf death was greater in control groups (11.06% and 10.73% in CPP and CM, respectively) compared with monitored cows (0.00% and 1.69% in MPP and MM, respectively). Multiparous cows with calf loss had increased relative risk (relative risk = 3.487) for early culling compared with multiparous counterparts with no neonatal loss. Daily milk production in the first 2 mo was 3.79 kg greater in multiparous cows with no dead calf, compared with their counterparts. A significant difference in median days open was found in MPP and CPP (118 and 148 d, respectively). In the final economic model, different simulations were analyzed. They were created assuming different prices or hypothesizing calving monitoring only in primiparous animals. The model estimated different, but always positive, net return. In conclusion, implementing a calving alarm system led to a net return from €37 to 90 per cow per year (€1 = US$1.15 at the time of the study). However, the device alone is not sufficient: it must be supported by qualified calving monitoring and assistance. Optimized personnel presence in the calving area at the right time leads to prompt calving and neonatal calf assistance and colostrum feeding within the first hours of life, thus reducing calf death and days open, and increasing milk production.

Buffalo breeding is common in Southern Italy. Dystocia compromises dam's and newborn health and welfare. Difficult parturition could be solved through prompt calving assistance, even if the identification of the beginning of delivery is challenging. Herein we aimed to evaluate a remote calving alarm system in 15 Mediterranean buffalo heifers. An intravaginal probe was placed close to the external cervical os once premonitory signs of delivery were observed. No vaginal discharge nor signs of discomfort were notified in the days following the insertion of the probe. Heifers calved from 48 to 72 hours after the alarm was activated. The system correctly warned the farm personnel at the beginning of stage II of parturition, except for 2 cases. In the former, the intravaginal probe was expelled but the poor carrier network coverage negatively affected phone's signal quality; in the latter, recurrent vaginal prolapse was responsible for non-retention of the probe. Overall median expulsive phase was 68 ± 8 minutes, while the expulsion of a female calf took 54 ± 22.0 minutes and 90 ± 34.0 minutes in males, with significant difference (P=0.02). Deliveries were homogeneously distributed across a 24-hour interval. No retention of fetal membranes nor metritis was identified at postpartum clinical examination. The calving alarm system used in this work was well tolerated in buffalo heifers. The introduction of smart technology in buffalo farming could contribute to the overall farm net return by reducing calf losses, especially for calves born from sexed-sorted semen, and by increasing animal welfare through quick resolution of dystocia. Further studies will be necessary to evaluate the net return in buffalo farms which will implement a remote calving alarm system on a wider population.

In vitro fertilization of oocytes in general is a process that is exposed to high oxygen concentration (20%) resulting in the...

Background Despite being one of the major causes of infertility in mares, the mechanisms responsible for equine endometrosis are still unclear and controversial. In the last few years, many investigations focused on local immune response modulation. Since it is generally accepted that endometrial fibrosis increases with age, we hypothesize that older mares could show altered local immune modulation, initiating a pro-inflammatory and tissue remodeling cascade of events that could lead to endometrosis. The aim of this study, indeed, is to evaluate and describe the local gene expression of genes involved in acute inflammatory response and fibrosis (COL1A1, COL3A1, TNFA, MMP9, IL6, TGFB1 and TGFBR1), together with others associated to immune modulation (DEFB4B, IDO1 and FOXP3), in uterine specimens from mares of different age. Results Twenty-five Standardbred mares were involved in the study with age ranging from 7 to 19 years (mean 10.40 ± 4.42). They were divided by age into two groups: G1 (n = 15, less than 10 years old) and G2 (N = 10, greater than 11 years old). Specimens from the uterus’ right horn-body junction were collected and processed for histology evaluation and RT-qPCR assay.Gene expression of DEFB4B, MMP9 and TNFA was higher in younger mares, suggesting a balance in immune modulation and tissue remodeling. Interleukin-6 and COL3A1 gene expressions were greater in older animals, probably indicating inflammatory pathways activation and fibrosis increase. Although no differences in fibrosis and inflammation distribution could be found with histological examination among G1 and G2, our results suggest a possible involvement of DEF4BB in regulating the local immune response in younger mare’s uterus (G1); age may contribute to the dis-regulation of DEFB4B transcription and, indirectly, influence the extracellular matrix homeostasis. Transcription of IDO1 and FOXP3 genes, instead, does not seem to be age related, or to be involved in local immune-response and tissue remodeling functions. Conclusions Further investigations are needed in order to clarify the interactions between the expression of DEFB4B, IL6, TNFA, COL3A1 and MMP9 and other local signals of immune-modulation and tissue remodeling, in mares in a prospective study design.

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 .05). The effect of breed was observed only for HTC that was higher in PRI than BS (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.

During the transition period, high-yielding dairy cows suffer from negative energy balance, intense lipomobilization and impaired lipid metabolism; this metabolic condition can lead to overburdened triglycerides accumulation in the liver, known as liver lipidosis, which has been associated to impaired fertility in dairy cows. The mechanisms of this impairment can be in principle correlated with the presence and the extent of ectopic fat depots. However, current methods for evaluating fat accumulation in liver and in the reproductive tract suffer from low resolution, sensitivity, and specificity. Confocal microscopes are equipped with Gallium arsenide phosphide detectors, thus enabling the acquisition of intense signals from tissue biopsies. This method could differentiate whether fat deposition occurred without requiring sample sectioning. Here, we examined with this technique liver, uterine and ovarian samples of heifers and regularly slaughtered repeat breeder and overconditioned dairy cows, to quantify lipid droplets and depots at a submicrometer scale with high specificity. With the aid of this technique, we found lipid depots in uterine and ovarian specimens. Moreover, we found that the size and number of depots increased with the degree of liver lipidosis. Further studies are needed to elucidate the relationship between the severity and extent of these deposits and the fertility of lactating dairy cows. Since tissues other than liver display different characteristic lipid droplet distributions, this technique can be potentially employed to shed new light on the pathogenesis of lipidosis and to assess new risk factors for infertility.

High-yielding dairy cows are metabolically challenged during transition, when intense mobilization and hepatic oxidation of lipids is achieved, thus leading to fatty infiltration, ketosis and generalized inflammation. The condition is associated to periparturient diseases and poor fertility. The aim of this study was to assess whether serum lipoprotein concentrations in the dry period could influence the occurrence of postpartum diseases and reproductive performance in dairy cows. The study was carried out on 30 multiparous Holstein Friesian cows. Blood samples were collected at dry-off (-60 days), 30 days after dry-off and within 12 h after parturition for biochemical and serum lipoprotein assays. From 10 to 60 days after parturition milk was collected twice weekly after feeding, for milk whey progesterone assay. The Optimal Cutpoint package identified a threshold of 89% for serum High Density Lipoprotein (HDL) concentration at the beginning of the dry period with 95% of confidence interval. Cows with serum HDL greater than 89% (High group, n = 10) showed better reproductive performance when compared to those with low values (Low group, n = 18). The odds ratio for reproductive disorders in High group was 0.6875, however, differences were not significant probably due to both the reduced number of animals per group and overall low incidence of postpartum reproductive disease. First postpartum luteal activity occurred around day 23, while the second one between days 40 and 48. The average calving to first AI interval was 64.00 ± 3.95 days and 94.50 ± 12.32 days in High and Low group, respectively (P 0.05). The calving-conception interval was 129.86 ± 24.42 days and 199.18 ± 24.73 days in High and Low groups, respectively (P 0.05). Low group displayed an increase in liver markers, that is total bilirubin, with 0.46 ± 0.09 mg/dL and 0.23 ± 0.08 mg/dL, in Low and High group respectively (P 0.05), and NEFA/cholesterol ratio, with 0.30 ± 0.06 and 0.14 ± 0.03, in Low and High groups, respectively (P 0.05), at parturition. Concentrations of HDL89% at dry-off could be suggestive of improved liver adaptation to the transition, and probably of enhanced fertility in High group.

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.

The aim of this study was to develop a system for the monitoring of calving to both reduce perinatal mortality and improve dairy cow fertility by preventing the majority of post-partum reproductive pathologies. Eighty dairy cows were assigned to the protocol of calving monitoring using GSM (Global System for Mobile Communications) technology. The application of GSM technology and the proper management of calving facilities comprise reliable approaches for calving assistance and improvements in reproductive efficiency and neonatal viability. Based on the results of this study, we advocate the use of GSM technology on large farms for intensive milk production.

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.

Gastrin (GAS) and insulin (INS) play a significant role in the regulation of feed intake, as well as in the homeorhetic adaptations to different physiological stages, nutrient supply and requirements. The aim of the study was to verify if plasma GAS and INS concentrations in adult does are affected by the physiological state, change in response to the meal and are correlated with feed intake and energy balance. Dry matter intake (DMI) and pre and post-prandial plasma concentrations of gastrin-17 and insulin were recorded in 21 female goats fed hay and different quantities of concentrate (maize grain) twice daily in different physiological states. DMI was lower during the week of oestrous activity, compared to the period of anoestrus – progressively decreasing during pregnancy and increasing during the post-partum period, without significant differences between early and late lactation. Plasma GAS concentrations were significantly affected by physiological status, being higher (P < 0.05) during lactation than during pregnancy. Overall plasma GAS concentrations increased (P < 0.01) from pre-feeding levels (169 ± 24 pg/ml), to 30 min after feed ingestion (185 ± 29 pg/ml). The increase occurred 10 and 20 min (P < 0.05) after feeding, regardless of the diet composition. Both pre- and post-prandial GAS concentrations were related to DMI (P < 0.001), but not to the energy intake. Plasma INS on the other hand was not significantly affected by the physiological status. The pre-feeding plasma INS concentration, across all physiological states, was 1.36 ± 0.32 ng/ml and increased (P < 0.01) to 1.45 ± 0.38 ng/ml 30 min after feeding. Pre-feeding INS, but not post-prandial INS, increased with increasing proportions of maize in the diet and energy intake (P < 0.05). Overall INS was negatively correlated (P < 0.05) to DMI. It was concluded that cephalic reflexes are efficient stimula for both GAS and INS release in adult goats fed twice daily. Plasma GAS concentrations were related to hay intake and total DMI, thus with the volume of ingesta and presumably the time spent eating. GAS concentrations were not affected by the type of feed, nor the energy intake or the flux into the abomasum. Pre-feeding plasma INS was mainly dependent on the energy intake, while the post prandial increase was not dependent on the size or quality of the meal, or on the energy intake. Dietary manipulation and feed ingestion can thus affect the circulatory levels of these two main endocrine factors.

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 (P0.05). 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.

Background Whether 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. Methods For 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. Results In 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. Conclusions Milk 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.

Nitrate (NO_ 3) and nitrite (NO_ 2) can be ingested with water and feeds and NO_ 2 can be produced from NO_ 3 by rumen and intestinal microbes. Moreover,NO_ 3 and NO_ 2 are endogenously produced by cells of various tissues from nitric oxide (NO) and arginine (Passi, 2000). Toxicity of NO_ 3 is casued by the reduction to NO_ 2 in the rumen environment. This ion competes with superoxide for hemoglobin (Doyle et al., 1982), producing methemoglobin (MtHb), which does not carry oxygen. The reaction is reversible, but the transient hypoxia can be exacerbated if the amount of ingested nitrates is high, causing fall of blood pressure and increase of heart rate........

Although semen quality is one of the major traits that influence breeding soundness examination outcomes in bulls, field conditions occasionally do not allow for the collection of semen samples by means of an artificial vagina. The aims of the present study were to report the results of a large number of semen collections that were performed via the transrectal massage (TRM) of the accessory sexual glands of Italian yearling beef bulls and compare this semen collection method to the artificial vagina (AV) method in term of breeding soundness examination outcomes; furthermore, we determined whether the breed affected the semen characteristics. In the TRM group (n = 475), the semen samples were collected via TRM of the accessory sexual glands, and in the AV group (n = 502), the AV method was used. In the TRM group, semen samples were obtained from 81.3% of the bulls and penile protrusion was observed in 87.6% of the animals during semen collection. The sperm concentrations (920.5 ± 439.0 vs. 281.0 ± 259.8 × 10(6)/mL) and the percentages of total abnormal spermatozoa (22.8 ± 15.0 vs. 18.8 ± 12.9) were significantly higher in the AV group than those in the TRM group. The percentage of bulls that did not meet the minimum requirement for normal cells (≥70%) was 6.2% higher in the AV group than that in the TRM group (P 0.05). Moreover, the samples collected from Chianina bulls by TRM exhibited a lower percentage of motile sperm and a higher percentage of abnormal spermatozoa when compared with the other two breeds. The major drawbacks of the TRM technique were the inability to conduct complete evaluation of the libido and mating ability of the yearling bulls, a significant reduction of the number of spermatozoa collected, and an increase in the variability of the semen characteristics due to breed. In conclusion, despite the drawbacks, TRM guarantees that semen evaluation can be conducted in cases in which the semen samples cannot be collected with the AV method.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein, which protects biomembranes from oxidative damages, and it also accounts for almost the entire selenium content of mammalian testis. The present investigation was performed to localise PHGPx in the testis and in epididymal and ejaculated spermatozoa of the bull by using light and electron immunomicroscopy. The study also aimed to further clarify the possible functions of the protein in bull fertility. In the testis, spermatogenic cells of the adluminal tubular compartment showed cytoplasmatic immunostaining; whereas, in the epididymal and ejaculated spermatozoa immunostaining was specifically localised at the level of the head and mid-piece. Ultrastructural data revealed the presence of signals for PHGPx in different subcellular compartments of maturing and mature sperm (mitochondria, chromatin, nuclear envelope, acrosomes, cytoskeletal structures) suggesting that this enzyme plays versatile and important biological roles during spermatogenesis. The final localisation of the immunostaining at acrosomal level puts forward a new role of the protein which further emphasises its relevance in male reproduction: it is reported to anchor substrate of the sperm acrosome to the oocyte zona pellucida during the fertilisation process.

The objective of this study was to characterize the glycoconjugates present in the zona pellucida of the follicular oocytes in sheep, goats and pigs. The zona pellucida was stained with periodic acid-Schiff, low iron diamine, high iron diamine, and nine different lectin horseradish conjugates: Con-A, SBA, DBA, PNA, RCA-I, GSA-II, WGA, LTA and UEA-I. Staining with DBA, PNA, SBA and RCA-I was performed with and without saponification with KOH and sialidase digestion. The results showed the presence of neutral and acidic glycoconjugates with different terminal sugars and also sialic acid radicals in the zona pellucida of all the animal studied. In particular, the positive staining with WGA, SBA, PNA and RCA-I suggests the presence of oligosaccharides with N-acetyl-D-glucosamine and sialic acid linked to the penultimate beta-N-acetyl-D-galactosamine and to the disaccharide galactosyl-(beta 1-3)-N-acetyl-D-galactosamine. The terminal trisaccharide sialic acid galactosyl-(beta 1-4)-N-acetyl-D-glucosamine was identified only in the zona pellucida of ovine and porcine oocytes. Thus, the zona pellucida exhibited species-specific variations in the content and distribution of lectin-binding patterns that may reflect the species specificity of gamete interaction.

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.

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.

The effect of a rapid variation of dietary starch and fat on metabolic and oxidative status in lactating ewes was investigated. Fifteen sheep at the end of pregnancy were assigned to three experimental groups and fed until 32 days from lambing a complete diet (22% starch and 3% fat on DM basis). At day 33, a group of ewes continued to receive the control diet (CTR), while the HST (high starch) group received a complete diet with 28% starch and 2.6% fat, and the HFA (high fat) group a diet with 16% starch and 5.4% fat, the diets were formulated to be iso-energetic. Blood and milk samples were collected before the change of diets (20 days), just after (35 days) and after 50 days from lambing. The HFA diet lead to a higher milk fat content compared to the CTR and HST diets, but the average daily gain of lambs during the trial was similar. The inclusion of starch in the diet increased blood glucose, insulin and lactate and reduced β-hydroxybutyrate (BHBA) and pH, while HFA reduced lactate and increased pH. Metabolic changes were associated with variations of markers of oxidative stress at 50 days. Total glutathione (GSx) decreased in the CTR and HST groups, while increased in HFA. Superoxide dismutase (SOD) activity in erythrocytes was higher only for the ewes fed HFA diet. Present data indicate that rapid modification of diet composition affects metabolic and oxidative homeostasis in lactating sheep.

Stress activates the hypothalamo–pituitary–adrenal axis leading to enhanced glucocorticoid secretion and concurrently disrupts ovarian cycle. Plant polyphenols are known to posses antioxidant and anti-inflammatory proprieties. This could be of interest for ovarian cycle when stressing conditions lead to progesterone enhancement and hamper normal reproduction activity. The present study examined whether ovarian follicular development and progesterone secretory pattern are affected by exogenous ACTH administration in heifers. Moreover, the effect of grape polyphenols in endometrium of heifers, under adrenocorticotropic hormone challenge, is evaluated in terms of transcriptional patterns of genes related to inflammation, oxidative stress and endometrial functions. At day 14 of synchronized estrous cycle, Holstein Friesian heifers received injections of either saline (CTR group) or adrenocorticotropic hormone (ACT group) agonist every 12 h for 7 days. Another group (POL group) of animals received the same treatment plus an oral supplementation of 15 g/day of grape skin extract. Cortisol and progesterone were analysed in the blood samples collected at days 0, 3, 6, 9, 12, 14, 17, 21, 24 of the estrous cycle. Endometrial biopsies were collected at diestrus (day 18) and at estrus and a panel of gene expressions were quantified by real-time PCR. ACTH administration increased both cortisol (P < 0.001) and progesterone concentrations (P < 0.01) compared to CTR group. PGHS-2 was significantly (P < 0.01) up-regulated in the POL group compared to ACT and CTR groups at diestrus and at estrus. FOXO3 and TIS11b were down-regulated in the CTR group compared to ACT and POL groups. The PGHS-2, SOD2 (P < 0.05), FOXO3 and TIS11b (P < 0.10) genes were down-regulated at estrus in all groups compared to diestrus. An interesting role of polyphenols in modulating the expression levels of PGHS-2 in endometrial tissue and on the activation of TIS11b and SOD2 through c-AMP-dependent signalling was suggested.

Growing evidence suggests that among the causes which deteriorate qualitative and functional characteristics of semen after freezing and thawing, there are those linked to oxidative stress and decrease detoxification of antioxidants present in the cells and seminal plasma. Overall, cryopreservation reduces the spermatozoal GSH content. In the present study we assessed the motility, capacitation status, ability to undergo acrosome reaction and levels of GSH in bovine spermatozoa after freezing and thawing, and compared the effects of the egg yolk tris-citrate and a commercial extenders (Bioxcell®). Six semen samples were collected from each of five bulls and frozen and thawed in the two extenders. The level of total GSH of bovine spermatozoa detected in the raw semen ranged from 246.5 to 776.2 pmoles/mg of protein. The Bioxcell® extender was superior in preserving GSH content (647.2 ± 300.1 versus 223.6 ± 154.0 pmoles/mL; P

The objectives of the present study were (i) to establish the mean value of scrotal circumference (SC), sperm motility, concentration and morphology at 13+/-1 months of age for Chianina, Marchigiana, and Romagnola breeds and (ii) to assign Italian beef bulls at the end of a growth performance test to a potential breeder category by applying the guidelines of the Society for Theriogenology in 1993 (SFT93). Of 1,315 bulls, 869 were not given the breeding soundness examination for the following reasons: not passing the growth performance test (n=445), no training for semen collection (n=404), and presence of genital abnormalities (n=20). Testicular length and diameter and SC exhibited a logarithmic trend over time, with an R(2) value of 0.963, 0.979, and 0.978 (P

The biochemical and molecular effects of tomato pomace (1.3% lycopene) or grape skin (87% polyphenols) supplementation for the control of hand-on induced stress in sheep were investigated. Twenty ewes were fed with a same basal diet for a preliminary period of 30 days and thus assigned to five groups: control group without any antioxidant (CTR); α-tocopherol (VIE, 100 mg/head/day); grape skin extracts (GRA, 5.5 g/head/day); tomato pomace (TOM, 10 g/head/day); tomato pomace and grape skins (TOG, 5.5 g/head/day of grape skins and 10 g/head/day of tomato pomace). Stress was administered by sampling blood at 0, 2, 3, 5, 7, 10 and 15 days after the beginning of the antioxidant administration. For the experiment, only samples at 0, 3 and 15 days were analysed. Plasma malondialdehyde (MDA), blood glutathione (GSx) and blood glutathione peroxidase (GPx) were not affected by dietary antioxidants, but significantly changed during the experimental period for all the sheep. A significant down-regulation of mRNA glutathione- S -transferase (GST) expression was observed 3 days after the addition of grape skins ( P = 0.006). After 15 days from the beginning of antioxidant administrations, the mRNA expression of cytochrome P450 (P450) showed a consistent reduction ( P = 0.011) in CTR, TOM and VIE groups, and superoxide dismutase (SOD) was up-regulated for TOG, GRA and TOM groups ( P = 0.000). Present results indicate that grape skins were more effective in inducing specific transcriptional activiy of genes involved in oxidant defences than tomato pomace and Vitamin E. The positive effect of Vitamin E is instead more evident in the short-term variations of GSx and GPx.

Granulosa-theca cell tumors (GTCTs) are able to secrete variable amounts of sex steroids and immunoreactive inhibin (ir-INH). Although the pituitary appears to be affected by the presence of a GTCT, pituitary responsiveness to exogenous GnRH has not been examined. The aims of the present study were to: (i) assess the plasma hormone concentrations of ir-INH, gonadotropins and sex steroids in eight mares with GTCT and (ii) assess the responsiveness of pituitary gonadotroph cells to exogenous GnRH stimulus both before and after tumor removal. In seven mares, the contralateral ovary was firm, small and inactive. Histopathological observations of the tumors confirmed the presumptive diagnosis of a GTCT. Four mares, judged to be in vernal transition period (n=2) and in the breeding season (n=2), were used as controls. A single intravenous injection of 40 microg of GnRH agonist was given to each mare and blood samples were collected every 15 min from 2 h before to 4 h after injection. In four GTCT mares, this procedure was repeated 20 (n=2) and 90 (n=2) days after tumors removal. All plasma samples were analyzed for concentrations of ir-INH, LH, FSH, estradiol-17beta (E2), testosterone (T) by RIA and progesterone (P) by EIA. Results showed that E2 levels were significantly higher (P0.001) in control animals compared to E2 levels in GTCT mares before and after surgery. P and T concentrations were not statistically different between the groups. Baseline levels of ir-INH were greater (P0.05) in GTCT mares before surgery than in control mares, and decreased to undetectable levels after neoplasia ablation. Baseline FSH did not differ between control and GTCT animals either before or after the ovaries were removed. LH baseline values appeared to be higher for affected mares, but the difference was not statistically significant. Maximum release (MR) and area under the gonadotrophin release curve (AUC) after the GnRH challenge for both the gonadotrophins were similar between the groups.

The imbalance between reactive oxygen species (ROS) and antioxidant capacity of the organism leads to a condition of oxidative stress (Urso and Clarkson, 2003). Studies in humans and laboratory animals have reported that oxidative stress is related to some common degenerative diseases, such as cancer and cardiovascular pathologies (Pellegrini et al., 2003). Oxidative stress has also been identified as causative agent for diseases, such as decline of immune function and atherosclerosis (Meydani et al., 1998). In particular, reactive oxygen metabolites such as superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.) have been reported to act as cytotoxic agents and damage unsaturated lipids in membranes (Girotti, 1998).

Several Mycoplasma species may adversely affect bovine spermatozoa viability and embryo development. Mycoplasma mycoides ssp. mycoides large-colony (LC) has been isolated from naturally aborted bovine fetuses and from bull semen. The objective of this study was to evaluate whether M. mycoides ssp. mycoides LC contaminated bovine ejaculates could (i) impair in vitro fertilizing ability of bull spermatozoa, (ii) impair embryo development, and (iii) evaluate potential spread by reproductive technologies. In the present study, spermatozoa of 10 fertile bulls were contaminated with M. mycoides ssp. mycoides LC, at a final concentration of 1.5 million CFU/ml and incubated for 60 min before evaluating spermatozoa motility and acrosome reaction inducibility with calcium ionophore. In addition, in vitro contaminated semen of a bull previously shown to have a good in vitro fertilizing ability, was used in an IVF procedure. Embryo development stage on Day-7 of culture was evaluated. Spermatozoa and embryos at morula and blastocyst stages were routinely processed for transmission electron microscopy observation. Both mean total and progressive motility decreased (P < 0.01 ) upon spermatozoa incubation with Mycoplasma. One-hour incubation with calcium ionophore increased the percentage of acrosome-reacted spermatozoa, although Mycoplasma contamination reduced calcium ionophore treatment efficacy (P < 0.05). Ultrastructurally, Mycoplasma microorganisms appeared as moderately electron-dense sphere-shaped particles, adhering to cell membranes. Sperm mid-piece sections showed numeric aberrations of the central singlets such as nine + zero or nine + one of the axonemal complex. Further morphological abnormalities included partial or total absence of dinein arms and radial fibers, with lack of the bridge and the central ring in 35.00 +/- 4.20% of contaminated cells, whereas these abnormalities were not observed in uninfected ones. The IVF trials showed that two-four cell blocks were higher (P < 0.05) in the infected group. Ultrastructure of Day-7 contaminated embryos showed Mycoplasma particles adhering and infiltrating the outer layer of the zona pellucida. Our investigations suggest that M. mycoides ssp. mycoides LC contaminating the bovine ejaculate induced adverse effects on in vitro spermatozoa-fertilizing ability and embryonic development. Some satisfactory quality transferable embryos could be produced in contaminated IVF systems. This could imply a potential transmission of this microorganism through reproductive technologies.

The study of cell turnover of mammary gland helps to gain understanding of the subtle modulation that largely determines milk production. To evaluate the effect of diet composition on cell turnover of mammary gland, pluriparous sheep were allotted to three experimental groups and fed from day 33 after lambing a control diet (BD), a high starch (HS) diet or a high fat (HF) diet. Biopsies of mammary gland tissue were collected at 50 days after lambing for in situ detection of cell death and RT-PCR analysis of bax, bcl-2, caspase-3 and GST expressions. Both apoptotic and proliferation indexes were significantly higher (P

The aim of the study was to compare the effects of heat stress and feed restriction on hormonal secretion (progesterone, oestradiol-17β, luteinizing hormone, follicle-stimulating hormone, prolactin and cortisol) in Holstein heifers. Ten pubertal heifers were divided into two groups of five (A and B) and housed in climatic chambers. After a pre-experimental period, the heifers were synchronised for oestrus and monitored for three (group B) or four (group A) consecutive oestrus cycles (OC). In the first OC, both groups were maintained under thermal comfort (TC) and fed on an ad libitum basis. In the second OC, group A was maintained under TC whereas group B was exposed to high air temperatures (HAT); both groups were fed on an ad libitum basis. In the third OC and until day 17 of the fourth OC, group A was kept under TC and fed a restricted diet (the same ration ingested by group B under HAT). At the end of HAT exposure, group B was removed from the study. Exposure to HAT caused development of ovarian cysts in two heifers, an increase in plasma concentrations of prolactin, a decrease in concentrations of cortisol and progesterone, and a 23% reduction in dry matter intake. Feed restriction did not modify any of the parameters considered. Results of this study indicated that the effects of HAT on the above parameters are not altered by a reduction in feed intake.

The testes of prepubertal and adult horses were investigated using 10 horseradish peroxidase conjugated lectins combined with sialidase digestion and potassium hydroxide treatment, to localise the oligosaccharide sequences of glycoconjugates during spermatid maturation. In adult animals, the lectins showed a variable affinity for spermatids and Sertoli cell apical extensions. Soybean agglutinin ( SBA ), peanut agglutinin ( PNA ), Ricinus communis agglutinin ( RCA - I ) and wheat germ agglutinin ( WGA ) bound to the acrosomal structures of spermatids, whereasGriffonia simplicifolia agglutinin ( GSA - II ) labelled these structures only during Golgi and cap phases. These results suggested that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides and that these carbohydrate chains undergo modifications during spermiogenesis. Sialic acid residues were not detected throughout the acrosomal development. The lectin binding pattern of Sertoli cells was very similar to that of acrosome of spermatids during the maturation phase. In sexually immature horses, only the degenerated germinal cells and the Leydig cells showed reactivity towards lectins. The first cells reacted with SBA and Dolichos biflorus agglutinin ( DBA ), the latter with SBA , PNA , WGA , GSA - II , Canavalia ensiformis agglutinin (ConA), Lens culinaris agglutinin ( LCA ) and also with DBA after sialidase digestion.

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.

Background Proper calving management of dairy herds is a crucial aspect of the bovine life cycle, as it has profound effects on calf viability and on the post-partum course of the dam. The objectives of this study were to monitor the calving process through the use of a remote alarm system and to determine the impact of prompt emergency obstetric procedures in case of dystocia for the prevention of stillbirths and post-partum reproductive pathologies, and for improving herd fertility. Six groups of experimental animals were studied: monitored heifers (n = 60) and multiparous cows (n = 60) were compared with non-observed animals (n = 60 heifers and n = 60 multiparous) giving birth during the same time period and housed in the calving barn, and with unmonitored animals placed in a dry zone (n = 240 heifers and n = 112 multiparous cows). Results The incidence of dystocia ranged from a minimum of 23.4% (monitored multiparous cows) to a maximum of 33.3% (monitored heifers), and there were no differences compared with control groups. However, the rate of stillbirth was higher in control groups than in the monitored groups (P P P P P P Conclusions The remote alarm system used to monitor the calving process assured the prompt presence of personnel, improving both the cow’s reproductive efficiency and neonatal viability.

The objective of this study was to investigate the effect of alpha-tocopherol deprivation on mammary gland involution and apoptosis in sheep. Two groups of four single lamb ewes were used. The control group received 100 mg/d of RRR-alpha-tocopherol supplementation and the experimental group received no vitamin E supplementation. After 3 mo of suckling, ewes were dried off, and blood samples from the jugular vein and tissue biopsies from the mammary gland were collected at d 1, 3, 5, and 8 after dry-off. The experimental group had lower plasma concentrations of alpha-tocopherol (1.8 vs. 4.2 micromol/L), lower glutathione peroxidase activity in erythrocytes, and higher concentration of malondialdehyde in plasma than the control group. Immunohistochemical analysis of tissue samples resulted in marked differences of bcl-2 and bax protein expressions during involution and between groups. The bax expression was decreased by alpha-tocopherol deprivation at 1, 3, and 5 d, but not at 8 d, while the bcl-2 score was higher only at 8 d (1.5 vs. 0.0 for experimental and control groups, respectively). As a result, the bcl-2 to bax ratios were increased for the experimental group at 1 and 8 d. During involution, apoptotic counts increased (from 0.12 to 4.06%), but no effects were detected in relation to bcl-2 to bax ratio and alpha-tocopherol. These results indicate that alpha-tocopherol can control bcl-2 expression, but not apoptosis in cells of the mammary gland during involution.