A comprehensive study of disorder of sex development in Staffordshire bull terrier dog

An 8‐month‐old female Staffordshire bull terrier was clinically examined because of external sexual organs abnormality—clitoral hypertrophy. As stated by the owner, the female dog had not been in heat yet. Serum profile of testosterone (3.39 ng/ml), as well as an anti‐M?llerian hormone (24.0 ng/ml), suggested the presence of testicular tissue. On the contrary, the estimated level of 17β‐oestradiol (24.6 pg/ml) was approximately two times higher when compared with the normal anoestrus values (5–10 pg/ml). A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicle or ovotestis (left) and hypoplastic testicle (right) was visible. Cranial portion of gonads was attached to structures indicative of bilateral epididymidis. The next tubular structures—oviducts were resected along with adherent parts of a hypoplastic uterus. Histological evaluation confirmed that the examined gonad samples were testicles with modified interstitial testicular tissue. Hypertrophy of interstitial space was predominantly formed by Leydig cells. Examination of a cross‐section through the head of suspected epididymidis confirmed their characteristic structures. In addition, the characteristic configuration of the oviducts was presented. The uterus consisted of three walls, in which the endometrium was hypoplastic with the presence of endometrial glands. No Y chromosome was detected by chromosomal analysis using CFA Y probe and the amplification of SRY‐gene coding region (813 bp) indicated genotype 78, XX; SRY‐negative. Sequencing of SOX9 gene exons 1–3 did not reveal any differences in exon 1 and 3. On the contrary, a few changes were determined in the SOX9 exon 2 sequences: G instead of A at position 103; C instead of reference T at position 115; GCG instead of reference CGC at position 138–140; T instead of reference C at positions 161, 164 and 167.     

Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles are Dose Dependent

The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross‐breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α‐MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non‐cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey′s and Dunnett′s tests) and chi‐square test (χ²). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.     

Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM⁺, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO₂ in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF‐1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF‐1 tested. Ultrastructurally, the non‐cultured control and IGF‐1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF‐1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.     

Immunohistochemical Localization of Fibroblast Growth Factor‐2 in the Sheep Ovary and its Effects on Pre‐antral Follicle Apoptosis and Development In Vitro

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF‐2 protein expression in ovine ovaries and to verify the effect of FGF‐2 on the morphology, apoptosis and growth of ovine pre‐antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α‐MEM⁺) alone or supplemented with FGF‐2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF‐2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF‐2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF‐2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF‐2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF‐2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF‐2 compared with the control medium and other FGF‐2 treatments. In conclusion, this study demonstrated the presence of FGF‐2 in ovine ovaries. Furthermore, 10 ng/ml FGF‐2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.     

Protein Localization of Epidermal Growth Factor in Sheep Ovaries and Improvement of Follicle Survival and Antrum Formation In Vitro

The aims of this study were to characterize EGF protein expression in ovine ovaries and to verify the effect of EGF on the in vitro development of isolated pre‐antral follicles. After collection, ovarian tissue was fixed for immunohistochemical analysis. Additional pairs of ovaries were collected, and secondary follicles were cultured for 18 days in α‐MEM⁺ (control) alone or supplemented with EGF (1, 10 or 50 ng/ml). The immunostaining for EGF was observed in oocytes from pre‐antral and antral follicles, in granulosa cells of primary and secondary follicles, as well as in cumulus and mural cells of antral follicles. After 18 days, the results showed that treatment with 50 ng/ml EGF significantly increased the percentage of morphologically normal follicles compared with the control group (α‐MEM⁺) and significantly reduced the precocious extrusion of oocytes and increased the percentage of antral follicles compared with the control and 1 ng/ml EGF. All the treatments induced a progressive and significant increase of the follicular diameter throughout the period of culture. However, there were no significant differences in follicular diameter or in the daily growth rate among treatments. In conclusion, this study demonstrated the presence of EGF in ovine ovaries. Moreover, 50 ng/ml EGF increased the percentage of normal follicles and improved antrum formation in isolated ovine follicles after 18 days of in vitro culture.     

Sex Steroid Levels in XY Males and Sex‐Reversed XX Males,of Rainbow Trout (Oncorhynchus mykiss), During the Reproductive Cycle

In this study, the annual cycle of the gonadal steroids testosterone (T), 11‐ketotestosterone (11‐KT), 17β‐oestradiol (E2) and 17α, 20β‐dihydroxy‐4‐pregnen‐3‐one (DHP) was determined using radioimmunoassay and then compared, for XY males (n = 35) and sex‐reversed XX males (n = 27) rainbow trout, to establish possible endocrinology differences. Both in XY males and sex‐reversed XX males, significant correlation was shown between body weight and T (r = 0.5046 and 0.34078, respectively; p < 0.0001) or KT (r = 0.52494 and 0.43545, respectively; p < 0.0001) concentrations. Plasma androgen levels in XY and sex‐reversed XX males were similar and showed an intense seasonal variation. The highest levels for T and 11‐KT were detected from December to April with a peak in January (51.67 ± 5.11 and 61.95 ± 4.25 ng/ml, for XY males and 57.1 ± 5.82 and 59.27 ± 4.84 ng/ml, respectively, for XX males). In addition, there was a positive correlation (p < 0.0001) between T and 11‐KT levels for XY males (r = 0.7533) and sex‐reversed XX males (r = 0.6019). Concentrations of DHP in XY males also showed seasonal variation with a peak in February (25.18 ± 12.99 ng/ml). However, DHP levels in sex‐reversed XX males were undetectable (<0.1 ng/ml) over the year. Levels of E2 were undetectable through the year in both groups of trout. In conclusion, the androgenic and oestrogenic profiles of sex‐reversed XX males were similar to those observed in XY males. The only difference in the annual gonadal steroid cycle between XY and sex‐reversed XX males was in the DHP profile.     

Growth differentiation factor‐9 improves development,mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles

This study analysed the effect of growth differentiation factor‐9 (GDF‐9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α‐MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α‐MEM⁺—control medium) or α‐MEM⁺ supplemented with 1, 10, 50 or 100 ng/ml GDF‐9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF‐9 than other treatments, except for 10 ng/ml of GDF‐9 (p > 0.05). Treatment containing 100 ng/ml GDF‐9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF‐9 showed more oocytes in MI than α‐MEM⁺, 1 or 50 ng/ml GDF‐9 (p < 0.05). In conclusion, 100 ng/ml GDF‐9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.     

The development and integrity of equine pre‐antral follicles cultured in vitro with follicle‐stimulating hormone (FSH) supplementation

This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre‐antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre‐antral follicles, the groups with 100 ng/ml FSH of 2‐days culture as well as 50, 100 and 200 ng/ml FSH of 6‐days culture provided the best results. In conclusion, the in vitro culture of abattoir‐derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.     

Anti‐Muellerian hormone concentration in bitches with histopathologically diagnosed ovarian tumours and cysts

Increased concentrations of Anti‐Muellerian hormone (AMH) can indicate a granulosa cell tumour as shown in women, mares and cows. To investigate AMH to differentiate canine granulosa cell tumour from other ovarian pathologies, we evaluated the ovaries of 63 bitches. Blood serum samples were collected before surgery for AMH analysis. Ovaries were submitted for histopathological examination. Fourteen bitches showed normal ovaries. These bitches had AMH values between 0.12 and 0.99 ng/ml. In 20 bitches ovarian cysts i.e., follicular cysts (n = 8), corpora lutea cysts (n = 7), subsurface cysts (n = 5) were diagnosed. These dogs had AMH values of 0.11–2.09 ng/ml. Bitches with small luteinized follicular cysts had slightly higher AMH values than those without ovarian alteration. In 29 cases ovarian neoplasms i.e., granulosa cell tumour (n = 9), epithelial tumours (n = 16), dysgerminomas (n = 3) and one sarcoma were identified. Anti‐Muellerian hormone values of bitches with an ovarian neoplasm except granulosa cell tumour ranged from 0.18 to 1.18 ng/ml. The AMH values of bitches with granulosa cell tumour ranged from 1.12 to ≤23 ng/ml and were significantly higher (p < .05) than in all of the other bitches. The cut‐off of 0.99 ng/ml gave a sensitivity of 100% and a specificity of 94.44% to diagnose granulosa cell tumour. In conclusion, markedly elevated AMH concentrations in bitches are indicative for a granulosa cell tumour. However, negative testing does not rule out the existence of small one. Differentiation of GCT from luteinized follicular cysts may especially be difficult.     

Bone Morphogenetic Protein‐6 (BMP‐6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre‐antral Follicles Cultured In Vitro

BMP‐6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP‐6) and recombinant follicle‐stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP‐6 (Experiment 2). Secondary follicles were cultured in αMEM⁺ alone (control medium) or supplemented with BMP‐6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP‐6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP‐6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non‐cultured control and after in vitro culture (MEM and 1 ng/ml BMP‐6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP‐6 at 1 ng/ml treatment. In conclusion, the low BMP‐6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.     

Diagnostic opportunities for evaluation of the exposure of dairy cows to the mycotoxins deoxynivalenol (DON) and zearalenone (ZEN): reliability of blood plasma,bile and follicular fluid as indicators

To investigate the usefulness of follicular fluid (FF) in relation to blood plasma and bile as indicators of exposure of dairy cows to ZEN, DON and their metabolites, a dose–response study was performed with 30 dairy cows. The cows, 10 in each group (named CON; FUS‐50, FUS‐100), received a diet with three different concentrations of Fusarium toxin‐contaminated maize. Thereby, the following dietary concentration were reached: CON (0.02 mg ZEN and 0.07 mg DON, per kg dry matter, DM), FUS‐50 (0.33 mg ZEN and 2.62 mg DON, per kg DM) and FUS‐100 (0.66 mg ZEN and 5.24 mg DON, per kg DM). ZEN, DON and de‐epoxy‐DON (de‐DON) were detected in FF. Based on the linear regression between toxin concentration in plasma and FF, it seems that about 50 % (m = 0.5) of ZEN present in plasma is present in FF while an increase of 1 ng/ml DON or de‐DON in plasma is paralleled by an increase of 1.5 ng/ml DON or 1.1 ng/ml de‐DON in FF. ZEN, DON and their metabolites, except zearalenone (ZAN), were also detected in bile. Contrary to DON and de‐DON, ZEN and its metabolites were accumulated in bile so that the concentration of ZEN and metabolites was much higher than for DON and de‐DON. The main compound was β‐zearalenol (β‐ZEL). The biliary ZEN, α‐zearalenol (α‐ZEL) and β‐ZEL concentration correlated linearly with each other with an uncertainty of <15 % (r² ≥ 0.86), whereas the ratio between ZEN: α‐ZEL: β‐ZEL was about 1.5:1:11. With the help of established linear relationship between toxin intake and toxin concentration, bile could be used as diagnostic indicator to assess the exposure of cows.     

Pharmacokinetics and bioavailability of oral firocoxib in adult,mixed‐breed goats

Pharmacokinetic (PK) studies of oral firocoxib in large animal species have been limited to horses, preruminating calves, and adult camels. The aim of this study was to describe pharmacokinetics and bioavailability of firocoxib in adult goats. Ten healthy adult goats were administered 0.5 mg/kg firocoxib intravenously (i.v.) and per os (p.o.) in a randomized, crossover study. Plasma firocoxib concentrations were measured over a 96‐hr period for each treatment using HPLC and mass spectrometry, and PK analysis was performed. The p.o. formulation reached mean peak plasma concentration of 139 ng/ml (range: 87–196 ng/ml) in 0.77 hr (0.25–2.00 hr), and half‐life was 21.51 hr (10.21–48.32 hr). Mean bioavailability was 71% (51%–82%), indicative of adequate gastrointestinal absorption of firocoxib. There were no negative effects observed in any animal, and all blood work values remained within or very near reference range at the study's conclusion. Results indicate that oral firocoxib is well‐absorbed and rapidly reaches peak plasma concentrations, although the concentration also decreased quickly prior to the terminal phase. The prolonged half‐life may suggest tissue accumulation and higher plasma concentrations over time, depending on dosing schedule. Further studies to determine tissue residue depletion, pharmacodynamics, and therapeutic concentrations of firocoxib in goats are necessary.     

Lack of glucuronidation products of trans‐resveratrol in plasma and urine of cats

Resveratrol has generated interest in cats due to reported health benefits. Cats have low activity of β‐glucuronidase, and we hypothesized they could not form two common resveratrol metabolites, resveratrol‐3‐O‐glucuronide and resveratrol‐4′‐O‐glucuronide. Resveratrol, 3 mg/cat/day, was given orally to intact male (n = 5) and female cats (n = 5) for 4 weeks. A control group (8 intact males) was used for comparison. Plasma and urine were collected weekly and analysed using high‐pressure liquid chromatography coupled with tandem mass spectrometry. Resveratrol and resveratrol‐3‐O‐sulphate, but no glucuronide metabolites, were detected in plasma and urine. Median (range 10–90th percentile) plasma resveratrol for control and treatment groups was 0.46 ng/ml (0.02–1.74 ng/ml) and 0.96 ng/ml (0.65–3.21 ng/ml). Median (range) plasma resveratrol‐3‐O‐sulphate for control and treatment groups was 6.32 ng/ml (2.55–10.29 ng/ml) and 11.45 ng/ml (1.47–53.29 ng/ml). Plasma resveratrol differed from control in week 4, while plasma resveratrol‐3‐O‐sulphate was different in all weeks (p < 0.05). Median (range) urine resveratrol for control and treatment groups was 0.28 ng/ml (0.05–1.59 ng/ml) and 19.98 ng/ml (8.44–87.54 ng/ml). Median (range) urine resveratrol‐3‐O‐sulphate for control and treatment groups was 26.71 ng/ml (10.50–75.58 ng/ml) and 108.69 ng/ml (11.83–231.05 ng/ml). All time points for urine resveratrol and resveratrol‐3‐O‐sulphate were significantly different from control (p < 0.05), except for weeks 1, 3 and 4 for resveratrol. The results support our hypothesis that cats are unlikely able to glucuronidate resveratrol, most likely due to a reduction in the activity of β‐glucuronidase.     

Insulin‐Like Growth Factor‐1 Regulates the Expression of Luteinizing Hormone Receptor and Steroid Production in Bovine Granulosa Cells

Luteinizing hormone LH plays important roles in follicular maturation and ovulation. The effects of LH are mediated by LH receptor (LHR) in the ovary. However, the factors that regulate the expression of LHR in bovine granulosa cells (GCs) are not well known. Insulin‐like growth factor‐1 (IGF‐1) is known to play a key role in the acquisition and maintenance of functional dominance. To better understand the roles of LHR expression and IGF‐1, we conducted three experiments to determine (i) mRNA expression of LHR in the GCs of developing follicles, (ii) the effects of IGF‐1 on LHR mRNA expression in cultured GCs and (iii) the effects of IGF‐1 on estradiol (E2), progesterone (P4) and androstenedione (A4) production by non‐luteinized GCs. In experiment 1, small follicles (<6 mm Ø) expressed lower levels of LHR than mid‐sized follicles (6–8 mm Ø) and large follicles (≥9 mm Ø) expressed the highest levels of LHR mRNA (p < 0.05). In experiment 2, IGF‐1 (1 and 100 ng/ml) increased (p < 0.05) the expression of LHR mRNA in GCs from small and large follicles. In experiment 3, IGF‐1 (0.1–100 ng/ml) increased A4 and E2 in GCs from both small and large follicles but increased P4 only in large follicles. IGF‐1 in combination with LH (0.1 and 1 ng/ml) increased P4 and A4 in large follicles, and increased E2 and A4 in GCs of small follicles. These findings strongly support the concept that IGF‐1 upregulates LHR mRNA expression as well as A4 and E2 production in GCs and that IGF‐1 is required for determining which follicle becomes dominant and acquires ovulatory capacity.     

Endocrinological characterization of an ovarian sex cord–stromal tumor with a Sertoli cell pattern in a Japanese Black cow

A Japanese Black cow was evaluated for prolonged post‐partum anestrus and enlargement of the right ovary. Transrectal ultrasonography revealed that the right ovary was markedly enlarged and had a solid appearance, while the left ovary was small and inactive. The presumptive diagnosis was directed towards granulosa‐theca cell tumour (GTCT) which was supported by markedly elevated plasma anti‐Müllerian hormone (AMH; 332.0 ng/ml), oestradiol (E₂; 103.3 pg/ml) and immunoreactive inhibin (ir‐INH; 2.1 ng/ml) in comparison with the diagnostic cut‐off points for bovine GTCTs. Since the cow had been infertile and had swelling of the udder, slaughter was chosen. Histopathological examination revealed that the tumour was an ovarian sex cord–stromal tumour (SCST) with a Sertoli cell pattern. These findings suggest that plasma AMH, ir‐INH and E₂ could be possible biomarkers for bovine ovarian SCST with a Sertoli cell pattern, whereas this case could not be distinguished from GTCTs based on endocrinological profile.     

Pharmacokinetics of the novel COX‐2 selective inhibitor vitacoxib in cats: The effects of feeding and dose

The purpose of this study was to determine the pharmacokinetics and dose‐scaling model of vitacoxib in either fed or fasted cats following either oral or intravenous administration. The concentration of the drug was quantified by UPLC‐MS/MS on plasma samples. Relevant parameters were described using noncompartmental analysis (WinNonlin 6.4 software). Vitacoxib is relatively slowly absorbed and eliminated after oral administration (2 mg/kg body weight), with a T_max of approximately 4.7 hr. The feeding state of the cat was a statistically significant covariate for both area under the concentration versus time curve (AUC) and mean absorption time (MAT_fed). The absolute bioavailability (F) of vitacoxib (2 mg/kg body weight) after oral administration (fed) was 72.5%, which is higher than that in fasted cats (F = 50.6%). Following intravenous administration (2 mg/kg body weight), Vd (ml/kg) was 1,264.34 ± 343.63 ml/kg and Cl (ml kg^?1 hr^?1) was 95.22 ± 23.53 ml kg^?1 hr^?1. Plasma concentrations scaled linearly with dose, with C_max (ng/ml) of 352.30 ± 63.42, 750.26 ± 435.54, and 936.97 ± 231.27 ng/ml after doses of 1, 2, and 4 mg/kg body weight, respectively. No significant undesirable behavioral effects were noted throughout the duration of the study.     

Anti‐Mullerian Hormone Concentration and Antral Ovarian Follicle Population in Murrah Heifers Compared to Holstein and Gyr Kept Under the Same Management

This study was performed to evaluate plasma concentrations of anti‐Mullerian hormone (AMH) and the ovarian antral follicle population (AFP) in different genetic groups. Cyclic heifers (13 Bubalus bubalis [Murrah]; 15 Bos taurus [Holstein] and 10 Bos indicus [Gyr]) were maintained under the same management and were synchronized with two doses of 150 μg IM d‐cloprostenol administered 14 days apart. After the second d‐cloprostenol treatment, heifers had their ovaries scanned daily by ultrasound to define the day of ovulation. On the same day, the AFP was determined and a plasma sample was collected to measure AMH. Murrah heifers had less AFP (25.6 ± 2.1 follicles; p = 0.01) and plasma AMH concentration (0.18 ± 0.03 ng/ml; p < 0.001) than Gyr (60.0 ± 12.2 follicles and 0.60 ± 0.12 ng/ml of AMH); however, data were similar when compared to Holstein (35.9 ± 6.8 follicles and 0.24 ± 0.06 ng/ml of AMH) heifers. Regardless of genetic background, there was a positive relationship between the AFP and plasmatic AMH concentration (Murrah [r = 0.62; p < 0.01], Holstein [r = 0.66; p < 0.001] and Gyr [r = 0.88; p < 0.001]). Also, when heifers were classified according to high‐ or low‐AMH concentration based on the average within each genetic group, high‐AMH heifers had greater (p < 0.0001) AFP than low‐AMH heifers. In conclusion, both Murrah and Holstein heifers presented lower plasma AMH concentration and AFP when compared to Gyr.     

Blood Metabolite Profiles in Cycling and Non‐cycling Friesian–Sanga Cross‐bred Cows Grazing Natural Pasture During the Post‐partum Period

An experiment was conducted to investigate the effect of plasma concentrations of the metabolic hormones [Growth hormone (GH), insulin and insulin‐like growth factor –I (IGF‐I)] and nutritional metabolites (Glucose, cholesterol, total protein, albumin, globulin, urea and creatinine) on the resumption of post‐partum ovarian activity in sixteen Friesian–Sanga cows grazing extensively on native grassland. Blood samples were taken from cows from week 1 to 16 post‐partum. Cows were classified as having resumed ovarian activity when a plasma progesterone concentration of ≥ 1.0 ng/ml was recorded for two consecutive weekly samples. Based on the resumption of ovarian activity, cows were classified as early‐cycling, late‐cycling or non‐cycling. The concentrations of the metabolic hormones were measured from week 1 to 10, while those of the nutritional metabolites were measured during week 1, 3, 5, 7 and 9 during the study period. The concentrations of the metabolic hormones, GH and insulin were similar (p > 0.05) in the three ovarian activity groups, likewise the concentrations of the nutritional metabolites, glucose, total protein, globulin, urea and creatinine. Plasma IGF‐I concentration was higher (p < 0.001) in early‐cycling (18.7 ± 0.74 ng/ml) than in late‐cycling (12.4 ± 0.75 ng/ml) and non‐cycling (10.4 ± 0.91 ng/ml) cows. Plasma cholesterol concentrations were significantly lower (p < 0.05) in early‐cycling (1.94 ± 0.15 mmol/l) compared with late‐cycling (2.48 ± 0.12 mmol/l) and non‐cycling (2.61 ± 0.11 mmol/l) cows. For plasma albumin concentrations, the levels recorded for early‐cycling cows were higher (40.7 ± 2.85 g/l) than in late‐cycling (34.4 ± 1.97 g/l) and non‐cycling (33.6 ± 2.66) cows. The results suggest that cows with lower plasma concentrations of IGF‐I and albumin, but higher plasma cholesterol concentrations were at risk of delayed resumption of post‐partum ovarian activity.     

The preliminary study on the effects of growth hormone and insulin‐like growth factor‐I on κ‐casein synthesis in bovine mammary epithelial cells in vitro

The effects of growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) on protein synthesis and gene expression of κ‐casein in bovine mammary epithelial cell in vitro were studied. The treatments were designed as follows: the growth medium without serum was set as the control group, while the treatments were medium supplemented with GH (100 ng/ml), IGF‐I (100 ng/ml), and GH (100 ng/ml) + IGF‐I (100 ng/ml). The quantity of κ‐casein protein was measured by ELISA, and the κ‐casein gene (CSN3) expression was examined by real‐time quantitative PCR (RT‐qPCR). Compared with the control group, all the experimental groups had greater (p < 0.05) expression of CSN3. The concentration of κ‐casein followed a similar response as CSN3, but the difference between the treatments and the control was not statistically significant (p > 0.05). Furthermore, no synergistic effect of GH and IGF‐I was observed for both the κ‐casein concentration and CSN3 expression. It is therefore concluded that GH or IGF‐I can independently promote the expression of CSN3 in bovine mammary epithelial cells in vitro.     

Correlation of Blood Flow of the Preovulatory Follicle to its Diameter and Endocrine Profile in Dairy Buffalo

Blood flow of the preovulatory follicle (POF) wall can be used as a predictor of the quality of POF. Our aim was to determine the correlation of blood flow of POF with the POF diameter, and intra‐follicular and plasma concentrations of Insulin‐like Growth Factor‐I (IGF‐1) and oestradiol in dairy buffalo. Nine Murrah buffalo subjected to an ovulation synchronization protocol (Ovsynch) were assessed on day 10 of the protocol for diameter and blood flow of POF, followed by the aspiration of follicle fluid. Prior to follicular aspiration, blood samples were obtained from jugular vein for estimation of IGF‐1 and oestradiol. The vascularity of POF was determined (Range: 250–967 pixel²) along with intra‐follicular and plasma concentration of IGF‐1 (Range: 9.3–31.8 ng/ml and 14.7–29.7 ng/ml respectively) and oestradiol (Range: 124.2–447.9 ng/ml and 0.25–1.05 ng/ml respectively). Diameter of the POF was weakly correlated (r = 0.21, p < 0.01) with blood flow to it. As compared to POF diameter, the blood flow of POF had greater positive correlation with intra‐follicular and plasma concentrations of hormones (IGF‐1 and oestradiol). A strong positive correlation was recorded between intra‐follicular IGF‐1 and oestradiol. Also, plasma concentrations of oestradiol and progesterone were negatively correlated In brief, assessment of the blood flow of the POF is a non‐invasive and reliable indicator of its functional competence as compared to the POF diameter.