Acid–Base Parameters and Steroid Concentrations in Pre‐Ovulatory Follicles and Plasma of Lactating Dairy Cows with Spontaneous and Synchronized Oestrus or Follicular Cyst

The study aimed to compare the acid–base balance and steroid concentrations between follicular fluids (FF) of pre‐ovulatory follicles derived from a spontaneous oestrus (SO), synchronized or induced oestrus (IO) and follicular cysts (CYS) and between FF and blood in dairy cows. Forty‐two dairy cows were included in this study. The animals were allocated to three groups: SO (n = 23); IO (n = 11) using GnRH at day 0 and day 9 and PGF₂α at day 7; and animals with CYS (n = 10). The follicular fluids (FF) were aspirated from the cyst/pre‐ovulatory follicles (? ≥ 15 mm) after SO and after second GnRH dose in IO by transvaginal ultrasound‐guided ovum pick‐up technique. Blood samples (BL) were collected in heparinized vacutainer tubes. The oxygen tension (pO2) in FF of IO was higher (p < 0.05) than in SO and CYS groups. There were negative correlations (p < 0.001, r = ?0.89) between FF and blood pO2. The carbon dioxide tension (pCO2) and lactate level in FF of CYS group were higher (p < 0.05) than in SO and IO groups. There were negative correlations (p < 0.01, r = ?0.73) between blood and FF pO2. Oestradiol‐17β concentration in pre‐ovulatory follicles and plasma of the SO group was higher (p < 0.001) than in IO and CYS groups. Progesterone concentration in pre‐ovulatory follicles and plasma of the SO and IO groups was lower (p < 0.01) than in CYS group. Plasma androstenedione concentration in SO and IO groups was higher (p < 0.05) than in CYS group. In conclusion, acid–base parameters, E2 and P4 levels in the follicular fluid of both IO and CYS groups were deviated greatly from the physiological level (disturbances of intrafollicular/intracystic environment), which may affect the quality of both the oocyte and the granulosa cells.     

Porcine Luteal Function in Relation to IGF-1 Levels Following Ovulation During Lactation or After Weaning

Contents This study presents relationships between peripheral progesterone and Insulin-like Growth Factor-1 (IGF-1) concentrations during the early luteal phase in sows. Data were derived from three experiments, one with primiparous weaned sows (n = 21) and two with multiparous sows that either ovulated during lactation (n = 23) or after weaning (n = 12). The sows that ovulated during lactation did so due to an intermittent suckling regime (inhibition of suckling for 12 h each day from day 14 of lactation) or due to treatment with PG600. IGF-1 concentrations varied considerably among experiments, and were the lowest in the multiparous sows, regardless of whether they were weaned or lactating: 68 ± 5 and 85 ± 8 ng/ml in the two experiments with multiparous sows vs 188 ± 15 ng/ml in the primiparous sows. Progesterone concentrations were lowest for the lactating sows. Overall, the increase in progesterone during the early luteal phase was strongly correlated with IGF-1 concentrations (r = 0.7). However, the correlation was low in multiparous lactating sows (r = 0.28; p < 0.10) and nonsignificant in multiparous weaned sows (r = 0). The weaned multiparous sows had IGF-1 levels comparable to lactating multiparous sows, but higher progesterone levels. In conclusion, these data show a positive relationship between peripheral IGF-1 and progesterone concentrations in vivo during the early luteal phase. In lactating sows, IGF-1 concentrations are probably a limiting factor for progesterone secretion, although other factors may be involved.     

Pre‐Selection Test to Identify High Responder Donor Goats

The aim of the study was to evaluate the feasibility of pre‐selection of high or low responder does prior to the superovulatory protocols. Twenty Saanen does received 800 IU of equine chorionic gonadotropin (eCG) at the end of long‐term progestogen treatment. Fourteen days later, a second progestogen protocol associated with a multiple‐dose follicle stimulation hormone (FSH) treatment (5 IU/kg of FSH, in six decreasing doses between days 4 to 6 of the protocol) was administered. Transrectal ultrasound was used to assess the follicular status at the beginning of superovulatory treatments, at the oestrous onset and on the seventh day of the oestrous cycle for counting corpora lutea (CL). A significant lower number of CL was obtained in eCG‐treated in comparision with FSH‐treated does (p < 0.05). A quartic regression was able to explain the relationship between the number of CL in response to both treatments (r²=0.50; p < 0.05). Seventy per cent (14 of 20) of does maintained the same ovulatory response (high or low) after treatments. The Kappa (κ = 0.40; p < 0.05) and Spearman (rs = 0.39; p = 0.08) coefficients were able to show a relationship between treatments. Regarding the follicular status, there is a significant relationship between the number of small follicles (r = 0.71; r²=0.47; p < 0.01) and total follicles (r = 0.60; p < 0.01) at eCG and first FSH dose with the number of CL. Moreover, it was found a negative relationship between the presence of large follicles and the number of CL in response to eCG treatment (r = ?0.44; p < 0.05), but not from FSH (p > 0.05). In conclusion, the screening test with eCG has the potential to identify Saanen does that will better respond to the superovulatory protocol with FSH. In addition, it highlighted the importance of an ultrasound evaluation prior to the beginning of superovulatory treatments with FSH to characterize the follicular status and identify the potential donors of high ovulatory response in MOET programmes in goats.     

Assessing the usefulness of B‐mode and colour Doppler sonography,and measurements of circulating progesterone concentrations for determining ovarian responses in superovulated ewes

The main goal of this study was to assess the usefulness of two imaging modalities, namely the B‐mode and colour Doppler sonography, and serum progesterone (P₄) concentrations for determining the ovarian response in superovulated ewes. Twenty‐four sexually mature Santa Inês ewes underwent the superovulatory treatment consisting of eight injections of porcine FSH (total dose of 200 or 133 or 100 mg; n  =  8 ewes/total dose) given at 12‐hr intervals and initiated 48 hr before CIDR ^® (Pfizer Inc., Auckland, New Zealand) removal. Six days after natural mating, the ovaries of all donor ewes were visualized and examined with transrectal ultrasonography and then with videolaparoscopy to identify and enumerate corpora lutea (CL ) and luteinized unovulated follicles (LUF s). Jugular blood samples were collected just prior to ovarian examinations. The total number of CL (r  =  .78 and 0.83, p  <  .0001) and LUF s (r  =  .74 and 0.90, p  <  .0001) enumerated using the B‐mode and colour Doppler ultrasonographic technique, respectively, were correlated with that ascertained by videolaparoscopy. Circulating concentrations of P₄ were related directly to the number of healthy CL (r  =  .73, p  =  .0002) and inversely to the number of prematurely regressing CL (r  = ?.46, p  =  .03), but the accuracy of predicting the number of short‐lived CL with serum P₄ concentrations was very poor. The present results indicate that ultrasonographic imaging and serum P₄ measurements on the day of embryo recovery are useful indicators of total/normal CL numbers and both ultrasonographic techniques can be used to quantify LUF s in superovulated ewes.     

Formation of Corpora Lutea and Central Luteal Cavities and Their Relationship with Plasma Progesterone Levels and Other Metabolic Parameters in Dairy Cattle

The corpus luteum (CL) may be looked upon as a compact or cavitary structure. A number of papers have addressed the relationship between CL type and parameters such as fertility or progesterone levels. The present study assessed the morphological and functional sequence observed in cows with different CL types, comparing pre-ovulatory follicle size, progesterone levels, luteal tissue formation and some blood biochemical parameters (calcium, albumin, inorganic phosphorus, glucose, magnesium, copper and zinc), oestrus cycle length and oestrus expression, as a function of CL type. Twenty-eight lactating dairy cows from two commercial dairy farms in southern Spain were studied. Oestrus detection was performed by monitoring daily oestrus behaviour, and artificial insemination (AI) was perfomed using the AM/PM rule. Ovaries and uterus were sonographically examined and blood samples were collected to measure progesterone and various biochemical parameters. There was a slight tendency towards the appearance of luteal cavities when pre-ovulatory follicles were larger (1.9 ± 0.2 vs 1.7 ± 03; p = 0.074). Fertility was not affected by cavity presence (cavity = 42.9% and compact = 57.1%; n.s.). Luteal tissue and function were not modified as a function of CL type. Cows with luteal cavities displayed significantly higher levels of albumin, suggesting a possible metabolic influence on the formation of these structures, although specific research is required to confirm this observation.     

Effect of hCG in the Presence of hCG Antibodies on the Follicle, Hormone Concentrations, and Oocyte in Mares

Follicle blood flow, follicular-fluid and plasma hormone concentrations, and oocyte quality were studied 30 h after an ovulation-inducing hCG treatment when the pre-ovulatory follicle was 32 mm. Mares were grouped as positive (n = 16) and negative (n = 44) for hCG antibodies before the experimental hCG treatment. Percentage of the follicle wall with blood flow signals was less (p < 0.05) in the antibody positive group than in the negative group. The concentrations of follicular-fluid oestradiol and free IGF1, and plasma oestradiol were greater (p < 0.001), and follicular-fluid progesterone (p < 0.001) and plasma LH (p < 0.02) were less in the antibody-positive group than in the negative group. For recovered oocytes at 30 h (n = 37), the antibody-positive group had fewer (p < 0.001) mature (MII) oocytes than the antibody-negative group. Results were attributable to highly effective neutralization of the hCG in the antibody-positive group.     

Effects of Exogenous Tumour Necrosis Factor-α on the Secretory Function of the Bovine Reproductive Tract Depend on Tumour Necrosis Factor-α Concentrations

The aim of study was to correlate tumour necrosis factor-α (TNF) infused doses used with the TNF concentrations achieved and with the secretory function of both the ovary and the uterus in cows. We evaluated the concentrations of progesterone (P4), prostaglandin (PG)F_2α, PGE₂ nitric oxide (NO) and TNF in the jugular vein and vena cava caudalis as parameters of exogenous TNF action on the female reproductive tract. Aortae abdominalis of cows (n = 18) were infused with saline or two doses of TNF (luteolytic – 1 μg or luteotrophic – 10 μg). In the peripheral blood, 1 μg TNF concentrations achieved within the range of 30–45 pg/ml, and 10 μg TNF provoked a sharp increase in achieved concentrations at a range of 250–450 pg/mL). The TNF concentrations achieved in vena cava caudalis were five to six times higher than that in peripheral blood (p < 0.001). One microgram TNF increased PGF_2α and NO (p < 0.001) and decreased P4 (p < 0.05). The higher TNF dose stimulated P4 and PGE₂ (p < 0.01). TNF infusion at luteolytic dose achieved its concentrations at the physiological range previously observed in cows. Luteotrophic TNF dose achieved the concentrations in vena cava caudalis that are much higher than physiological level and were previously noted in pathological circumstances (i.e. mastitis , metritis ).     

Metabolic load in dairy cows kept in herbage‐based feeding systems and suitability of potential markers for compromised well‐being

Herbage feeding with only little input of concentrates plays an important role in milk production in grassland dominated countries like Switzerland. The objective was to investigate the effects of a solely herbage‐based diet and level of milk production on performance, and variables related to the metabolic, endocrine and inflammatory status to estimate the stress imposed on dairy cows. Twenty‐five multiparous Holstein cows were divided into a control (C+, n = 13) and a treatment group (C?, n = 12), according to their previous lactation yield (4679–10 808 kg) from week 3 ante partum until week 8 post‐partum (p.p.). While C+ received fresh herbage plus additional concentrate, no concentrate was fed to C? throughout the experiment. Within C+ and C?, the median of the preceding lactation yields (7752 kg) was used to split cows into a high (HYC+, HYC?)‐ and low‐yielding (LYC+, LYC?) groups. Throughout the study, HYC+ had a higher milk yield (35.9 kg/d) compared to the other subgroups (27.2–31.7 kg/d, p < 0.05). Plasma glucose (3.51 vs. 3.72 mmol/l) and IGF‐1 (66.0 vs. 78.9ng/mL) concentrations were lower in HYC?/LYC? compared to HYC+/LYC+ cows (p < 0.05). Plasma FFA and BHBA concentrations were dramatically elevated in HYC? (1.1 and 1.6 mmol/l) compared to all other subgroups (mean values: 0.5 and 0.6 mmol/l, p < 0.05). Saliva cortisol, plasma concentrations of serum amyloid A (SAA), haptoglobin (Hp), beta‐endorphin (BE) and activity of alkaline phosphatase (AP) were not different between C+ and C?. In conclusion, herbage‐fed high‐yielding cows without supplementary concentrate experienced a high metabolic load resulting in a reduced performance compared to cows of similar potential fed accordingly. Low‐yielding cows performed well without concentrate supplementation. Interestingly, the selected markers for inflammation and stress such as cortisol, Hp, SAA, BE and AP gave no indication for the metabolic load being translated into compromised well‐being.     

Effects of Short-Term High Carbohydrate or Fat Intakes on Leptin, Growth Hormone and Luteinizing Hormone Secretions in Prepubertal Fat-Tailed Tuj Lambs

The aim of the current study was to evaluate the effects of high carbohydrate or fat diets, fed for 15 days at the end of breeding season, on leptin, GH and LH secretions in prepubertal fat‐tailed Tuj lambs. For that purpose, 9‐month‐old ram‐lambs were divided into three groups as control group (fed with basal ration, n = 4), high carbohydrate (HC) group, basal ration plus barley, n = 4), or high fat (HF) group (basal ration plus by‐pass fat, n = 4). For the measurement of leptin and GH, blood plasma samples were collected on days 1, 4, 9 and 14 of the experiment. For the measurement of LH pulse frequency, serial blood samples were collected every 15 min for 6 h on day 14. Lambs were weighed and body condition scored (BCS) on days 1 and 15. Body weight and BCS increased towards the end of the study (p < 0.05). The BCS was higher in high energy groups at the end of the experiment (p < 0.05). Diet affected plasma leptin concentrations (p = 0.002) but time did not. The GH concentrations were not affected by diet or time. The LH pulse frequency appeared to be higher in HC and HF groups but there were no statistical difference between the groups. There was a significant positive relationship between overall BCS and corresponding leptin concentrations (R² = 0.263; p = 0.010) and between LH pulse frequency and leptin concentrations (R² = 0.594; p = 0.003). In conclusion, the present study suggests that rather than type of energy, amount of energy intake and body energy reserves are much important regulators of plasma leptin concentrations and LH pulse frequency in fat‐tailed Tuj lambs.     

Comparison of the Effect of the Aromatase Inhibitor, Anastrazole, to the Antioestrogen, Tamoxifen Citrate, on Canine Prostate and Semen

This study compared the efficiency of the aromatase inhibitor, anastrazole, with the antioestrogenic receptor blocker, tamoxifen, on normal (NRL) and hyperplastic prostate glands. Forty healthy dogs were classified as NRL (n = 18) or abnormal (ABN) with benign prostate hyperplasia (n = 22). The dogs were randomly assigned to one of the following six groups, treated for 60 days; oral placebo for normal (NRL-PLC; n = 6) and abnormal (ABN-PLC; n = 6), oral anastrazole 0.25–1 mg/day, for normal (NRL-ANZ, n = 6) and abnormal (ABN-ANZ, n = 8) and oral tamoxifen citrate 2.5–10 mg/day for normal (NRL-TMX; n = 6) and abnormal (ABN-TMX; n = 8) dogs. The dogs were evaluated before treatment and then monthly for 4 months. At the end of the treatment, the prostatic volume decreased by 28.5 ± 4.3%, 21.6 ± 6.3% and 0.7 ± 1.0% in the ABN-TMX, ABN-ANZ and ABN-PLC (p < 0.01), respectively. From then on, prostatic volume began to increase without reaching pre-treatment values at the end of the study. In the ABN animals, there were no differences for this parameter between ANZ and TMX treatment (p > 0.1), whereas in the NRL animals ANZ produced a less pronounced decrease (p < 0.05), libido, testicular consistency and scrotal diameter decreased during treatment in the TMX group (p > 0.05). These parameters and sperm volume, count, motility and morphological abnormalities remained unaltered throughout the study in the ANZ and PLC groups (p > 0.05). There were no haematological nor biochemical side effects. Anastrazole might offer a safe and effective alternative for the medical management of dogs with benign prostatic hyperplasia.     

Comparison of Two Doses of the GnRH Antagonist, Acyline, for Pregnancy Termination in Bitches

Gonadotropin-releasing hormone (GnRH) antagonists are particularly useful when a rapid inhibitory effect on the gonadal axis is required. The aim of this study was to test the efficacy and clinical safety of a low and high dose of the third generation GnRH antagonist, acyline, on pregnancy termination in female dogs. The effect of the antagonist on the progesterone (P₄) serum concentration was also described. Twenty-one mid-pregnant bitches were randomly assigned to a single subcutaneous (SC) dose of a placebo (PLACE; n = 7), a low (ACY-L; 110 μg/kg; n = 6) or high (ACY-H; 330 μg/kg; n = 8) dose of acyline. The animals were followed up for 15 days. All ACY treated but no placebo-treated animals terminated their pregnancy by abortion (p < 0.01). The ACY-L and ACY-H groups interrupted their pregnancy 7 ± 1.9 and 6.4 ± 1.3   days after treatment, respectively (p = 0.7). A significant interaction between treatment and day was found (p < 0.01) for P₄ serum concentrations when PLACE was compared with both ACY groups. No difference was found for this hormone between both ACY groups (p > 0.05) where P₄ diminished throughout the study. The decreasing rate varied among animals and was closely related to the time of abortion when P₄ reached basal concentrations. In PLACE animals, gestation progressed normally and P₄ did not change throughout the study (p > 0.05). None of the bitches presented side effects. It was concluded that acyline safely terminated mid-pregnancy by permanently decreasing P₄ serum concentrations.     

Effect of Estradiol and Progesterone on Metabolic Biomarkers in Healthy Bitches

The aim of this study was to evaluate the possible association between hormonal changes that occur during oestrus and biomarkers related with glucose metabolism (glucose and insulin), lipid metabolism (lipidic profile and BChE) and adipokines (adiponectin and ghrelin) in healthy bitches. For this purpose, we measured these analytes in serum of bitches, at two times: before (T1) and after (T2) the LH peak that were established according to progesterone concentrations. Increased levels of total cholesterol (p < 0.01), high density lipoprotein cholesterol (HDL‐C) (p < 0.01), low density lipoprotein cholesterol (LDL‐C) (p < 0.01), adiponectin (p < 0.01) and ghrelin (p < 0.05) were observed at T2 in comparison with T1. No statistically significant changes were observed in serum glucose, insulin, homoeostasis model assessment for insulin sensitivity (HOMA), triglycerides and BChE. When all data of T1 and T2 were pooled, serum adiponectin showed positive correlation with progesterone (r = 0.353; p = 0.022) and HDL‐C (r = 0.307; p = 0.048), and negative with insulin (r = ?0.429; p = 0.005), HOMA (r = ?0.446; p = 0.003) and BChE (r = ?0.522; p < 0.001). Ghrelin showed negative correlation with estradiol (r = ?0.701; p = 0.004). BChE was negatively correlated with estradiol (r = ?0.441; p = 0.018) and glucose (r = ?0.343; p = 0.028), and positively with insulin (r = 0.460; p = 0.003) and HOMA (r = 0.505; p < 0.001). In conclusion, changes in metabolic biomarkers occur in bitches after LH peak, characterized by increased lipids (total cholesterol, HDL cholesterol and LDL cholesterol) without changes in BChE activity, and increased adiponectin and ghrelin concentrations, without significant changes in glucose and insulin.     

An Experimental Model to Study Resistance Index and Systolic/Diastolic Ratio of Uterine Arteries in Adverse Canine Pregnancy Outcome

The aim of this study was to describe the changes in the resistance index (RI) and systolic/diastolic ratio ( S / D ) of the uterine arteries during mid-pregnancy abortion induction in the dog. Sixteen 30–35 day pregnant bitches were randomly assigned to either a pharmacological protocol to interrupt gestation (n = 8) or were used as untreated control group (n = 8). Doppler assessments of uterine arteries blood flow were carried out before the initiation of the protocol and then every other day up to abortion (treated group) or parturition (control group). All treated bitches aborted 6 ± 1.2 days after initiation of the treatment (while none of the non-treated bitches aborted). Pre-treatment RI and S / D did not differ between groups (p > 0.2) while average post-treatment indexes were (mean ± SD): 0.62 ± 0.1 vs 0.53 ± 0.1 (p < 0.01) and 2.96 ± 0.9 vs 2.23 ± 0.3 (p = 0.01), for the treated and non-treated group respectively. Correlations between days to abortion and RI or S / D were 0.75 (p < 0.01) and 0.79 (p < 0.01) and, −0.78 (p < 0.01) and −0.73 (p < 0.01) for the treated and non-treated groups respectively. In the treated group, correlations between serum progesterone (P₄) concentrations and RI and S / D were −0.76 (p < 0.01) and −0.59 (p < 0.01) respectively. It is concluded that, during induction of abortion, RI and S / D of uterine arteries progressively increased while P₄ decreased.     

The Comet assay for detection of DNA damage in canine sperm

Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno‐, terato‐ or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A—median: 31.55%, interquartile range (IQR): 30.18–38.01; group B—median: 0.90%, IQR: 0.65–1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p < .001). There was a negative correlation between progressive motility and oxidative damage (r = ?.792; p = .001). Basal and oxidative DNA damage of spermatozoa are increased in dogs with non‐normospermic semen. In conclusion, and considering the elevated correlation with classical tests of sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs.     

Prevalence of Potentially Pathogenic Bacteria as Genital Pathogens in Dairy Cattle

Bacteria on the genital mucosa have been studied less in healthy, non-puerperal cows than in cows with puerperal endometritis. We have thus analysed bacteria in swabs from the vagina and cervix of post-puerperal cattle (n = 644). Out of the animals, 6.8% had aborted within the last 12 months, 2.6% and 11.6% showed signs of vaginitis and endometritis, respectively. In 17.2% of cervical swabs pathogenic gram-positive and in 11.5% pathogenic gram-negative bacteria were found. Arcanobacterium pyogenes was isolated from 41.3% of cows with endometritis and from 3.5% without endometritis (p < 0.05). From 12.5% of cows with abortion but from no cow without abortion, Staphylococcus aureus was recovered (p < 0.05). Out of 383 vaginal swabs, 88.3% were positive. In 3.4% of swabs pathogenic gram-positive and in 16.7% pathogenic gram-negative microorganisms were found. The percentage of positive vaginal swabs did not differ between pregnant and non-pregnant animals. In the genital tract, the percentage of swabs positive for normal mucosal bacteria decreased from caudally to cranially (p < 0.05). Pathogenic bacteria were found more often in cervical than in vaginal swabs (p < 0.05). In conclusion, bacteria on the vaginal and cervical mucosa in cattle involve a wide range of species. In animals without endometritis or vaginitis, colonization of the mucosa rather than infection has to be assumed.     

Improved Detection of Reproductive Status in Dairy Cows Using Milk Progesterone Measurements

This study tested a model for predicting reproductive status from in-line milk progesterone `measurements. The model is that of Friggens and Chagunda [Theriogenology 64 (2005) 155]. Milk progesterone measurements (n = 55 036) representing 578 lactations from 380 cows were used to test the model. Two types of known oestrus were identified: (1) confirmed oestrus (at which insemination resulted in a confirmed pregnancy, n = 121) and (2) ratified oestrus (where the shape of the progesterone profile matched that of the average progesterone profile of a confirmed oestrus, n = 679). The model detected 99.2% of the confirmed oestruses. This included a number of cases (n = 16) where the smoothed progesterone did not decrease below 4 ng/ml. These cows had significantly greater concentrations of progesterone, both minimum and average, suggesting that between cow variation exists in the absolute level of the progesterone profile. Using ratified oestruses, model sensitivity was 93.3% and specificity was 93.7% for detection of oestrus. Examination of false positives showed that they were largely associated with low concentrations of progesterone, fluctuating around the 4 ng/ml threshold. The distribution of time from insemination until the model detected pregnancy failure had a median of 22 days post-insemination. In this test, the model was run using limited inputs, the potential benefits of including additional non-progesterone information were not evaluated. Despite this, the model performed at least as well as other oestrus detection systems.     

Expression of MHC-I and -II in Uterine Tissue from Early Pregnant Bitches

The aim of the study was to investigate the expression of major histocompatibility complex (MHC)-I and -II in uterine tissues from pregnant and non-pregnant bitches, taken at different time periods after mating. The pregnant bitches were ovariohysterectomized during the pre-implantation (group 1, n = 4), implantation (group 2, n = 7) and placentation stage (group 3, n = 7). Non-pregnant animals in diestrus served as controls (group 4, n = 7). The expression of MHC- I and -II in salpinx, apex, middle horn, corpus uteri and at implantation sites was investigated by immunohistochemistry as well as qualitative and quantitative RT-PCR; MHC-I mRNA was detected in all tissues and with quantitative RT-PCR, and no significant changes were detected until placentation. Immunohistologically, at the apex and corpus site, the average number of MHC-II positive cells increased from the pre-implantation to the post-implantation stage (apex: 1.54 ± 1.21 to 3.82 ± 2.93; corpus: 1.62 ± 1.9 to 5.04 ± 4.95; p < 0.05). The greatest numbers of MHC-II positive cells were observed at placentation sites (6.64 ± 5.9). In parallel, a marked increase in the relative mRNA expression of MHC-II in uterine tissues was assessed from the pre-implantation to the placentation stage (relative to Glycerinaldehyd-3-phosphate-Dehydrogenase (GAPDH): 6.9 ± 9.5, 8.4 ± 5.8, p > 0.05). Immunohistologically, in the salpinx, significantly greater numbers of MHC-II positive cells were found in the tissues of pregnant animals than in the control group (p < 0.05). It is proposed that the increase in MHC-II is pregnancy-related, even though the impact on maintenance of canine pregnancy is still unclear.     

Immunohistochemical study of Ki‐67 protein,androgen receptor,and estrogen receptor beta in testicular tissues of male pigs immunocastrated with different times of GnRH vaccination

This study aimed to investigate the immunoexpression of Ki‐67 protein, androgen receptor (AR), and estrogen receptor beta (ERβ) in testicular tissues of male pigs immunocastrated using GnRH vaccine (Improvac?, Zoetis Co., Ltd., Thailand) with different times. Totally, 30 male pigs were classified by castration protocol into three groups: T1 (n = 10) consisted of pigs immunocastrated at 14 and 18 weeks of age, T2 (n = 10) included pigs immunocastrated at 9 and 19 weeks of age, and C (n = 10) contained intact pigs. The results revealed that testicular length of pigs in C was longer than that of both T1 (8.1 ± 0.76 vs 6.5 ± 0.5 cm, p < 0.001) and T2 (8.1 ± 0.76 vs 6.9 ± 1.0, p = 0.007). Spearman correlation coefficients showed negative correlation between testicular length and H‐score of AR (r = ?0.38, p = 0.037), as well as positive correlation between testicular length and Ki‐67 index (r = 0.602, p < 0.001). Generally, mean Ki‐67 index and mean H‐scores of AR and ERβ of pigs in T1 were not different from those in T2 (p > 0.05). However, mean Ki‐67 index and mean AR H‐scores of T1 and T2 were significantly different from C group (p < 0.05). In summary, the immunocastration significantly affected testicular length, including expressions of Ki‐67, AR, and ERβ in pig testes. Moreover, the duration between two shots of GnRH vaccine could be extended from 4 to 10 weeks without difference in Ki‐67 protein, AR, and ERβ immunoexpressions.     

Accurate Ultrasonographic Prediction of Progesterone Concentrations Greater than 1 ng/ml in Holstein lactating dairy cows

To develop an ultrasonographic assay for determining plasma progesterone concentration (P₄) as < 1 ng/ml or ≥ 1 ng/ml, the corpus luteum (CL) area and P₄ were measured in 1094 multiparous Holstein cows. The area‐measuring function and frozen images were used to outline and measure CL imaged via ultrasonography, and CL area was estimated as a polygon of a continuation straight line. A significant correlation was found between CL area and P₄ (p < 0.001), and this analysis resulted in the following correlation equation: y = ?0.35 + 1.02x (r = 0.81). According to the correlation equation, a CL area of 1.3 cm² indicated a P₄ of 1 ng/ml. Based on this relationship, each animal was categorized into one of six groups, groups differed based on CL area, and the area ranges were as follows: < 1.3 cm² (Group A), 1.3–2.2 cm² (Group B), 2.3–3.2 cm² (Group C), 3.3–4.2 cm² (Group D), 4.3–5.2 cm² (Group E) and > 5.2 cm² (Group F). For each group, the proportion of cows whose P₄ was 1 ng/ml or more was 1.5% in Group A, 83.3% in Group B, 76.6% in Group C, 96.6% in Group D, 99.2% in Group E and 100% in Group F. There was a significant difference between Group A and the other five groups, and between Groups B or C and Groups D, E or F (p < 0.005). These results indicate that a functional CL does not exist when the CL area is less than 1.3 cm² and that it exists when the CL area is 3.3 cm² or more.     

Cefquinome Concentrations in Endometrium after Intrauterine Treatment of Cobactan 4.5%® in Mares and Inflammatory Response of the Endometrium to this Treatment

This study was conducted to measure the concentration of cefquinome in the endometrium of mares after intrauterine treatment and to evaluate associated inflammation. Mares (n = 14) were randomly assigned to one of the following groups: (i) control (n = 4) were either not treated (n = 2) or received (n = 2) lactated Ringer's intrauterine for 1 or 3 days; (ii) treated mares (n = 10) received intrauterine cefquinome for 1 or 3 days. After at least 10 days had passed following the last treatment and ovulation, mares were given Prostaglandin F2α (PGF2α) and were randomly assigned to an alternate treatment. Endometrial biopsy samples were taken at 2, 8, 24 and 48 h, or at 4, 12 and 36 h, after the last treatment. Biopsy samples were taken at the same time points from control mares (n = 2) and lactated Ringer-treated mares (n = 2). Cefquinome concentrations were quantified using a high-performance liquid chromatography (HPLC) assay and inflammation was assessed using haematoxylin and eosin (H&E)-stained sections. Concentrations of cefquinome [559 (1 day) and 595 μg/g (3 days) at 2 h, and 403 (1 day) and 370 μg/g (3 days) at 4 h] were similar between treatment groups at 2 and 4 h after treatment (p > 0.05). At 8 h, as well as at 24 and 48 h, concentrations were greater in the 3-day group (17 vs 301 μg/g, 3 vs 80 μg/g and 0.1 vs 0.2 μg/g, respectively) (p < 0.05). No significant differences (p > 0.05) in the inflammatory response at 2–48 h after treatment were found between groups.