Plasma concentrations of progesterone in the preceding estrous cycle are associated with the intensity of estrus and fertility of Holstein cows.

The aim of this study was to determine the association between concentrations of progesterone (P4) during previous the estrous cycle with the intensity of spontaneous or estrogen-induced estrous expression and pregnancy per artificial insemination (P/AI). A total of 1,953 AI events from lactating Holstein cows were used, consisting of 1,289 timed AI events from experiment 1 (Exp. 1) and 664 AI events from experiment 2 (Exp. 2). In Exp. 1, cows were bred after a timed AI protocol based on estradiol and P4. In Exp. 2 animals were bred upon spontaneous estrus detection. In both experiments cows were continuously monitored by an automated activity monitor (AAM), in Exp.1 a relative increase of activity was calculated (i.e., percentage of increase activity at estrus compared to cow's baseline activity) and in Exp.2, activity data from each cow were computed into an index value that ranged from 0 to 100. In Exp.2 duration (hours) of estrus were calculated and defined as the total time above the threshold (35 index). The intensity of estrous expression was determined for each event and classified as either high or low intensity using the median of each experiment. Blood samples were collected for P4 analysis in Exp. 1 at -4 d, 0 d, and 7 d relative to timed AI, and in Exp. 2 immediately following estrus (0 d), 7 d, 14 d, and 21 d post-AI. Concentration of P4 was classified as greater or lower according to the median value in each experiment. Cows with lower concentrations of P4 at AI had greater estrous expression in Exp. 1 (363.6 ± 5.2 vs. 275.9 ± 8.0% relative increase) and Exp. 2 (76.7 ± 1.9 vs. 67.4 ± 4.7 index; and 12.5 ± 0.5 vs. 9.3 ± 1.8 hours). Cows with a greater intensity of estrous expression at timed AI had greater concentrations of P4 at -4 d than cows with lower intensity estrus or no estrous expression (4.6 ± 0.2 vs. 3.6 ± 0.2 vs. 3.7 ± 0.2 ng/mL). Cows with greater concentrations of P4 at -4 d had greater P/AI (32.8 ± 4.4 vs. 22.4 ± 4.5%), whereas cows with lesser concentrations of P4 at d0 for either timed AI (35.2 ± 3.4 vs. 19.6 ± 4.6%) or spontaneous estrus (31.8 ± 2.8 vs. 23.4 ± 3.2%) had greater P/AI. Cows with greater concentrations of P4 7 d post-timed AI had greater P/AI compared with cows that had lower concentration of P4 (39.1 ± 2.9 vs. 24.7 ± 2.6%). Similarly, cows that had lower concentrations of P4 at 7 d, 14 d and 21 d post-spontaneous estrus tended to have lower P/AI when compared with cows with greater concentrations of P4. Overall, concentrations of P4 prior to and at AI were associated with greater estrous intensity and P/AI at both spontaneous and timed AI events.

Plasma metabolite changes in anestrous dairy cows with negative energy balance identified using 1H NMR technology / [Alterações do metabólito plasmático em vacas leiteiras com balanço energético negativo identificado usando a tecnologia 1H NMR]

The objective of the present study was to investigate the different plasma metabolites between anestrus and estrus postpartum dairy cows and to provide a theoretical basis for prevention of anestrus in dairy farm cows. In the experiment, one hundred and sixty-seven Holstein dairy cows were selected with similar age and parity. According to the concentration of ß-hydroxybutyric acid, non-esterified fatty acids and glucose in plasma during 14 to 21 days in milk, all dairy cows were determined as having a status of energy balance. According to the results of clinical symptom, rectal and B ultrasound examination at 60 to 90 days postpartum, these cows were divided into twenty estrus and twenty-four anestrus group, other dairy cows were removed. 1H nuclear magnetic resonance technology was utilized to detect the plasma metabolites changes and screen different plasma metabolites between anestrus and estrus cows. Ten different metabolites including alanine, glutamic acid, asparagine, creatine, choline, phosphocholine, glycerophosphocholine, low-density lipoprotein, and very-low-density lipoprotein were significantly decreased in anestrous cows compared with estrous cows. Metabolic pathway analyses indicated that differential metabolites were primarily involved in amino acid and glycerophospholipid metabolism. These metabolites and their enrichment pathways indicate that reduced steroid hormone synthesis precursors result in lower levels of estradiol and progesterone and cause anestrus in negative energy balance. These data provide a better understanding of the changes that may affect estrus of postpartum dairy cows at NEB status and lay the ground for further research.(AU)

O objetivo do presente estudo foi investigar os diferentes metabolitos do plasma entre o cio e o cio pós-parto de vacas leiteiras e fornecer uma base teórica para a prevenção do cio de vacas em fazendas de leite. No experimento, foram selecionadas 127 vacas leiteiras Holstein com idade e paridade similares. De acordo com a concentração de ß- ácido hidroxibutírico, ácidos graxos não esterificados e glicose no plasma entre 14 e 21 dias no leite, todas as vacas leiteiras foram determinadas em estado de equilíbrio energético. De acordo com os resultados dos sintomas clínicos, do exame de ultra-som retal e B aos 60 a 90 dias pós-parto, estas vacas foram divididas em vinte cios e vinte e quatro grupos de cio, outras vacas leiteiras foram removidas. A tecnologia de ressonância magnética nuclear 1H foi utilizada para detectar as alterações dos metabólitos plasmáticos e para triar diferentes metabólitos plasmáticos entre as vacas do cio e do cio. Dez diferentes metabólitos incluindo alanina, ácido glutâmico, asparagina, creatina, colina, fosfocholina, glicerofosfocolina, lipoproteína de baixa densidade e lipoproteína de muito baixa densidade foram significativamente diminuídos nas vacas antróficas em comparação com as vacas estro. As análises da via metabólica indicaram que os metabólitos diferenciais estavam principalmente envolvidos no metabolismo de aminoácidos e glicerofosfolipídios. Estes metabólitos e suas vias de enriquecimento indicam que a redução dos precursores da síntese de hormônios esteróides resulta em níveis mais baixos de estradiol e progesterona e causa anestros no balanço energético negativo. Estes dados fornecem uma melhor compreensão das mudanças que podem afetar o cio das vacas leiteiras pós-parto no estado de NEB e preparam o terreno para mais pesquisas.(AU)

Effects of blood pathological changes before TAI on pregnancy of dairy cows with anestrus and estrus / [Efeitos de mudanças patológicas de plasma antes de (TAI) na gestação de vacas em anestro e estro]

The objective of this study was to investigate the influence of plasma pathological changes before timed artificial insemination (TAI) on pregnancy of cows. The contents of estrogen (E2), progesterone (P4), glucose (Glu), selenium (Se), brain-derived neurotrophic factor (BDNF), and histamine (HIS) in plasma of 48 Holstein cows were measured before TAI. According to the estrus detection, the cows were divided into estrus (E) and anestrus (A) groups. After pregnancy testing at 28 d after TAI, two groups of E and A were divided into positive pregnancy of E group (EP+), negative pregnancy of E group (EP-), positive pregnancy of A group (AP+), and negative pregnancy of A group (AP-). The contents of E2, P4, Glu, Se, BDNF and hIS significantly differed among the four groups (P<0.01). The ROC analysis was used to determine the risk of negative pregnancy test (-) after TAI was increased when plasma E2 was less than 46.45 pmol/L in cows before TAI. The changes in E2, P4,hIS, Glu, and BDNF in the blood of natural estrus and natural anestrus cows affected the pregnancy after TAI. the level of E2 in plasma may be used to assess the risk of negative pregnancy after TAI.(AU)

O objetivo do presente estudo foi investigar a influência de mudanças patológicas de plasma antes de inseminação artificial (TAI) na gestação de vacas. O conteúdo de estrogênio (E2), progesterona (P4), glucose (Glu), selênio (Se), fator neurotrófico derivado do cérebro (BDNF), e histamina (HIS) no plasma de 48 vacas Holstein foi medido antes de TAI. De acordo com a detecção de estro, as vacas foram divididas em dois grupos: estro (E) e anestro (A). Após teste de gestação 28 d após TAI, dois grupos de E e A foram formados em gestação positiva do grupo E (EP+), gestação negativa do grupo E (EP-), gestação positiva do grupo A (AP+), e gestação negativa do grupo A (AP-). Os valores de E2, P4, Glu, Se, BDNF e hIS foram significativamente diferentes entre os quatro grupos (P<0,01). A análise ROC foi utilizada para determinar o risco de teste de gestação negativo (-) após aumento de TAI quando plasma E2 estava abaixo de 46,45 pmol/L em vacas antes de TAI. Alterações em E2, P4,hIS, Glu e BDNF no sangue de estro natural e anestro natural em vacas afetou a gestação após TAI. O nível de E2 no plasma pode ser usado para avaliar o risco de gestação negativa após TAI.(AU)

Short communication: Simultaneous measurements of estrus behavior and plasma concentrations of estradiol during estrus in lactating and nonlactating dairy cows.

Estrus is an important behavior that can potentially be subjected to genomic selection. Circulating estradiol concentrations at estrus may be a useful phenotype if the absolute concentrations of estradiol are associated with overt phenotypes for estrus (activity, rump touches, or both; e.g., mounts, chinrests) that can be easily observed. The objective was to measure plasma estradiol concentrations at estrus and associate these measurements with the increase in activity (steps per hour) and rump touches received at estrus. We also tested the effect of lactation on the estrus traits that we measured. Cows (n = 11 lactating and n = 9 nonlactating) were treated with PGF2α to synchronize estrus. A jugular vein was cannulated to collect blood every 2 h for plasma estradiol measurement. Plasma LH was measured during the periestrual period to determine the time of the LH surge. Cows were fitted with an accelerometer to measure activity (steps per hour) and a capacitive touch sensing device to measure the number of rump touches and total touch time. Plasma estradiol concentrations were poorly correlated with overt signs of estrus during the period leading up to maximum estrus activity. After peak estrus activity (when cows were going out of estrus and plasma estradiol concentrations were decreasing), a stronger correlation was detected between overt signs of estrus and plasma estradiol concentrations. Effective selection for improved estrus expression based on plasma estradiol concentrations will depend on whether the cow is coming into or going out of estrus at the time of blood sampling. An association existed between lactation and fewer number of hours in estrus when estrus was defined by an increase in activity (steps per hour). Lactating cows had a shorter interval from the onset of estrus to the LH surge, and the shorter interval to the LH surge may have reduced the period of elevated estradiol during estrus in the lactating cows. Understanding mechanisms that control the sensitivity of the cow to estradiol and making appropriate selection decisions based on these mechanisms will likely increase overt signs of estrus in dairy cows.

Sexual maturity, seasonal estrus, and gestation in female Indo-Pacific bottlenose dolphins Tursiops aduncus inferred from serum reproductive hormones.

Reproductive hormones in serum concentrations of progesterone, estradiol, and testosterone in female Indo-Pacific bottlenose dolphins (Tursiops aduncus, n = 12) housed in Ocean Park Hong Kong were investigated in the present study. Results showed that, onset of puberty of captive Indo-Pacific bottlenose dolphins was at 5 years while sexual maturity was at 6. Average serum progesterone concentrations in non-pregnant sexually mature individuals was 0.33 (0.25-0.97) ng/mL (interquartile), significantly higher than in immature ones 0.26 (0.25-0.38) ng/mL. This study found significant difference in serum estradiol concentrations between individuals at the onset of puberty (9.5 ± 1.7 pg/mL, ±SD) and not (below detection limit 9 pg/mL). A slightly seasonal breeding pattern, with progesterone values tend to be higher from February to October (0.38 [0.25-1.07] ng/mL) was inferred. During pregnancy, serum progesterone concentrations range from 10.54 ± 8.74 ng/mL (indexed month post-conception [IMPC] 0) to 25.49 ± 12.06 ng/mL (IMPC 2), and display a bimodal pattern with 2 peaks in early- (25.49 ± 12.06 ng/mL, IMPC 2) and late-pregnancy (21.71 ± 10.25 ng/mL, IMPC 12), respectively. Serum estradiol concentrations can seldom be detected in early-pregnancy and increase constantly in mid- (9.45 ± 1.83 pg/mL) and late-pregnancy (11.88 ± 3.81 pg/mL), with a spike (15.45 ± 6.78 pg/mL) 1 month prior to delivery. Serum testosterone concentrations elevate significantly in IMPC 7 (0.36 ± 0.10 ng/mL) compared to other months (0.16 ± 0.10 ng/mL) of the year. The present study provides normal concentration profiles for some reproductive hormones in female Indo-Pacific bottlenose dolphins and can contribute to the breeding monitoring of this species. Also, our study would shed further light on the reproductive physiology of small cetaceans.

Gut microbiota and blood metabolomics in weaning multiparous sows: Associations with oestrous.

This study was conducted to detect the potential relationship between changed plasma metabolites, intestinal microbiota and the weaning-to-oestrous interval in multiparous sows after weaning. Multiparous sows were allocated to two groups after weaning: the oestrous group (n = 15) with a weaning-to-oestrous interval ≤7 days or the anoestrous group (n = 15) with a weaning-to-oestrous interval >14 days. The levels of plasma reproductive hormones: oestradiol, follicle-stimulating hormone and luteinizing hormone, plasma total protein; blood urea nitrogen; cholesterol; high-density lipoprotein; and ammonia (NH3 ) were significantly lower in the anoestrous sows compared with the oestrous sows (p < .05). The plasma metabolomics analysis identified 14 metabolites (lactose, l-cysteine, cytosine, hydantoin, palmitoleic acid, arachidic acid, linoleic acid methyl ester, α-ketoglutaric acid, N(ε)-trimethyllysine, threo-ß-hydroxyaspartate, 3-(3-hydroxyphenyl) propionic acid and others) with lower concentrations and 12 metabolites (noradrenaline, 5-dihydrocortisone, p-cresol, 1,4-cyclohexanedione, 2,3-dimethylsuccinic acid and others) with higher concentrations in the anoestrous group compared with the oestrous group (p < .05). The 16S rRNA pyrosequencing analysis showed the relative increase in abundance of the Prevotella and the Bacteroides at the genus level in the anoestrous group (p < .05). At the phylum level, lower proportions of Firmicutes and Lentisphaerae were observed in the anoestrous group (p < .05). This study provided a comprehensive assessment of metabolic differences in the blood and differences in the gut microbiome composition between anoestrous and oestrous sows. And suggesting that this profiling approach may offer new insights into explaining the alteration of the gut microbiota and blood metabolomics are correlated with sex hormone secretion and the weaning-to-oestrous interval of sows after weaning.

Effects of Ovarian Transplant on Anti-Müllerian Hormone, Kisspeptin-1, and Kisspeptin-1 Receptor Levels in Rats.

OBJECTIVES: Information is scarce on levels of kisspeptin-1 and the kisspeptin-1 receptorin females after ovarian transplant. In this study, our aim was to explore serum estradiol, anti-Müllerian hormone, kisspeptin-1, and kisspeptin receptor levels, along with kisspeptin-1-positive cell density, in ovaries from rats after ovarian transplant. MATERIALS AND METHODS: For this study, 28 female Sprague Dawley rats were divided into 4 groups, with sham surgery performed on rats in group 1 (control group). Group 2 rats had ovaries transplanted under the peritoneum, and group 3 rats had their own ovaries transplanted subcutaneously. Group 4 rats were maintained in an estrous state. Serum anti-Müllerian hormone, kisspeptin-1, estradiol, and ovarian kisspeptin receptor levels were determined using commercial enzyme-linked immunosorbent assay kits. Kisspeptin-1-positive cell densities in the ovaries were determined immunohistochemically.The ovaries were also examined histopathologically. RESULTS: Our statistical analyses showed that levels of kisspeptin receptors in the ovaries were lowest in the subcutaneously transplanted group (group 3; 628.57 ± 35.69 pg/mL). The highest serum anti-Müllerian hormone levels were found in the estrous group (group 4; 16.91 ± 2.12 ng/mL). Differences between groups in terms of serum kisspeptin-1 and estradiol concentrations were not statistically significant. CONCLUSIONS: Our findings suggest that, in rats, results were better in the peritoneum transplant group than in the subcutaneous transplant group. We also found that serum anti-Müllerian hormone levels were lower in the transplant groups than in the estrous group, although levels were not completely decreased to zero. These results support the finding that ovarian activities continue after transplant.

Profile of bile acid subspecies is similar in blood and follicular fluid of cattle.

The composition of follicular fluid (FF) has an impact on the developmental capacity of the oocyte and the resulting embryo. FF is composed of blood plasma constituents which cross the blood follicular barrier and the secretory components of granulosa and theca cells. Moreover, it has been shown recently that follicular cells have the ability to synthesize bile acids (BAs). BAs are present in several fluids of mammals especially in bile, blood and urine. FF is an essential impacting factor on the oocyte quality and therefore resulting embryos. To achieve a better understanding of this subject, the presence and concentration of BAs were measured in fluid collected from bovine follicles, categorized according to their size, throughout two entire oestrus cycles and compared to those in blood and urine. The body fluids were collected during the same examination procedure and in total samples from four heifers were obtained. A broad spectrum of 11 BA derivatives was measured applying liquid chromatography-tandem mass spectrometry (LC-MS/MS). The simultaneous and direct quantification of BAs in different body fluids of cattle are reported. Within the follicular fluid, blood and urine, cholic acid and glycocholic acid are the dominant BA subspecies irrespective of the oestrus cycle stage. Moreover, BA concentrations in blood compared to those in the FF were similar. For the first time these results clearly highlight the presence of different BA subspecies in FF, blood and urine during the oestrus cycle in cattle.

Expression of the anti-Mullerian hormone, kisspeptin 1, and kisspeptin 1 receptor in polycystic ovary syndrome and controlled ovarian stimulation rat models.

Polycystic ovary syndrome represents a significant cause of female infertility. The aim of this study was to investigate the expression of anti-Mul-lerian hormone (AMH), kisspeptin 1 (KISS-1), and kisspeptin 1 receptor (KISS1r) in rat models of polycystic ovary syndrome (PCOS) and controlled ovarian stimulation (COS). For this purpose, 28 rats were assigned into four groups. Estrus and Diestrus groups consisted of rats in estrus and diestrus phases, respectively, while COS and PCOS groups consisted of rats with induced COS and PCOS, respectively. The serum AMH, KISS-1, and estradiol levels, and ovarian KISS1r levels were analyzed by enzyme-linked immunosorbent assay. Furthermore, histopathological analysis of the ovary tissue was done and ovarian KISS-1 expression was determined by immunohistochemical assay. The results revealed that ovarian KISS1r levels were higher in the Estrus (1271.43±51.98 pg/mL) and COS (1191.43±85.67 pg/mL) groups, compared to Diestrus and PCOS groups. The highest level of AMH was found in the Estrus group (16.91±2.12 ng/mL). The results indicate that AMH had no effect on the development of COS and PCOS, while KISS-1 was found to affect the development of COS in rats.

Screening for reproductive biomarkers in Bactrian camel via iTRAQ analysis of proteomes.

Bactrian camel is an ancient and precious species of livestock; that is, unique resources exist in the desert and have important economic and scientific value. In recent years, the number of Bactrian camels has declined sharply. Due to its long reproductive cycle and seasonal oestrus, the mechanism of oestrus is unknown. To identify candidate biomarkers of reproduction, we performed a comprehensive proteomic analysis of serum from Bactrian camel in oestrus and non-oestrus, using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with tandem mass spectrometry. We identified 359 proteins, of which 32 were differentially expressed: 11 were up-regulated and 21 were down-regulated in samples from camels in oestrus. We validated the differential expression of a subset of these proteins using qPCR and Western blot. Gene ontology annotation identified that the differentially expressed proteins function in cellular processes, metabolic processes and immune system processes. Notably, five of the differentially expressed proteins, PCGF5, histone H1.2, RBP4, FOLR1 and ANTXR2, are involved in reproductive regulatory processes in other animals. KEGG enrichment analysis demonstrated significant enrichment in several cardiac-related pathways, such as 'dilated cardiomyopathy', 'hypertrophic cardiomyopathy', 'cardiac muscle contraction' and 'adrenergic signalling in cardiomyopathy'. Our results suggest that candidate biomarker (PCGF5, histone H1.2, RBP4, FOLR1 and ANTXR2) discovery can aid in understanding reproduction in Bactrian camels. We conclude that the profiling of serum proteomes, followed by the measurement of selected proteins using more targeted methods, offers a promising approach for studying mechanisms of oestrus.

Comparison of progesterone assay by chemiluminescence or radioimmunoassay for clinical decision-making in canine reproduction.

The Coat-A-Count® radioimmunoassay has been long and widely used to determine the concentration of progesterone in serum or plasma of bitches (progRIA), but was discontinued in 2014. The Immulite® 1000 LKPG1 chemiluminescence immunoassay has gained prominence since 2003 to determine the concentration of progesterone in serum of bitches, but the assay changed in 2012 (Immulite® 1000 LKPW1). This study assessed the feasibility of using Immulite® 1000 LKPW1 (progImm) to estimate the time of clinically relevant events during oestrus and compared progRIA and progImm 2 and 3 days after the first or only day of the luteinizing hormone surge (LH1). ProgImm first exceeded 5.1 nmol/L on the same day that progRIA first exceeded 6 nmol/L, a proxy for the occurrence of the LH surge, or the day before in 28 of 31 (90%) of oestrous periods. ProgImm first exceeded 13.6 nmol/L on the same day that progRIA first exceeded 16 nmol/L (a proxy for the day of ovulation) or the day before in 34 of 35 (97%) oestrous periods. ProgImm first exceeded 5.4 nmol/L on LH1 or the day before in 24 of 25 (95%) of oestrous periods. The median of progImm 2 days after LH1 was 1.2 nmol/L lower than the 10.7 nmol/L of progRIA (p = 0.001). The mean of progImm 3 days after LH1 was 2.2 nmol/L lower than the 19.0 nmol/L of progRIA (p 0.001). In conclusion, the days on which progImm first exceeded 5.1 nmol/L, 13.6 nmol/L and 5.4 nmol/L effectively estimate the days on which progRIA reached 6 nmol/L or 16 nmol/L or LH1.

Comparative evaluation of DNA integrity using sperm chromatin structure assay and Sperm-Ovis-Halomax during in vitro capacitation of cryopreserved ram spermatozoa.

This study aimed to compare the ability of sperm chromatin structure assay (SCSA® ) and Sperm-Ovis-Halomax® to detect DNA fragmentation in frozen-thawed ram spermatozoa incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with oestrous sheep serum (SOF-ESS) at multiple time points (0-240 min). Incubation in SOF-ESS had no significant effects on SCSA® parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm-Ovis-Halomax® increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques, suggesting that SCSA® and Sperm-Ovis-Halomax® may quantify different types of DNA damage in ram spermatozoa under these experimental conditions.

Plasma Protein Comparison between Dairy Cows with Inactive Ovaries and Estrus.

To screen differentially expressed proteins in the blood dairy cows with inactive ovaries caused by a negative energy balance and to determine the roles of the identified proteins in the development of inactive ovaries.Holstein cows at 14 to 21 days postpartum in an intensive dairy farm were examined for their energy balance (EB) status by blood ß-hydroxybutyrate (BHBA) and assigned to the inactive ovary (IO) group (n = 50) and the normal oestrus control (CON) group (n = 50) at 60 to 90 days postpartum by means of the oestrus manifestation, rectal examination and B-ultrasound examination. Fourteen differentially expressed proteins from 61 proteins in the plasma of dairy cows with IOs were identified by iTRAQ/LC-MS/MS and GO, KEGG, and PATHWAY analysis. Eleven expressed proteins were upregulated, and 3 expressed proteins were downregulated. Among the 10 differentially expressed proteins verified by Western blot or ELISA, the relative expression levels of ALDOB, IGFBP2, ITIH3 and LDHB in mixed samples and single samples were consistent with the proteomic protein results. PKM2, GPX3, ALDOB, RBP4 and AHSG were significantly different between the two groups (P < 0.05); APOA4 and SPAM1 were not significantly different (P > 0.05) but were still downregulated in the ovarian resting group. This study confirmed that 14 plasma differential proteins in the inactive ovaries of postpartum dairy cows were associated with follicular development, and these findings provide a foundation for further research on the mechanism and prevention of inactive ovaries in dairy cows.

Menstrual blood derived mesenchymal stem cells combined with Bushen Tiaochong recipe improved chemotherapy-induced premature ovarian failure in mice by inhibiting GADD45b expression in the cell cycle pathway.

BACKGROUND: To investigate the therapeutic effects of menstrual blood derived mesenchymal stem cells (MB-MSCs) combined with Bushen Tiaochong recipe (BSTCR) on epirubicin induced premature ovarian failure (POF) in mice. METHODS: Twenty-four female C57BL/6 mice of 6-8 weeks were intraperitoneally injected with epirubicin to induce POF, and then they were randomized into 4 groups of 6 mice each and treated with PBS, MB-MSCs, BSTCR, and MB-MSCs combined with BSTCR, respectively. Six mice of the same age were used as controls. Vaginal smear, TUNEL and hematoxylin-eosin staining were to observe estrous cycles, ovarian cell apoptosis and follicles. Enzyme-linked immunosorbent analysis determined serum estradiol, follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH) levels. RT-qPCR and Western Blot analysis were to determine GADD45b, CyclinB1, CDC2 and pCDC2 expressions. RESULTS: Epirubicin treatment resulted in a decrease in the number of primordial, primary, secondary and antral follicles, an increase in the number of atretic follicles and ovarian cell apoptosis, a decrease in estradiol and AMH levels, an increase in FSH levels, and estrous cycle arrest. However, MB-MSCs combined with BSTCR rescued epirubicin induced POF through down-regulating GADD45b and pCDC2 expressions, and up-regulating CyclinB1 and CDC2 expressions. The combined treatment showed better therapeutic efficacy than BSTCR or MB-MSCs alone. CONCLUSIONS: MB-MSCs combined with BSTCR improved the ovarian function of epirubicin induced POF mice, which might be related to the inhibition of GADD45b expression and the promotion of CyclinB1 and CDC2 expressions. The combined treatment had better therapeutic efficacy than BSTCR or MB-MSCs alone.

Extended lactation in high-yielding dairy cows. I. Effects on reproductive measurements.

The objective of this prospective field study was to evaluate the effects of extending the lactation period on various reproductive measurements of high-yielding Holstein cows. On 40 d in milk (DIM), cows were gynecologically examined (transrectal palpation, sonography, vaginoscopy). Cows without signs of clinical endometritis were blocked by parity and were randomly allocated to 1 of 3 experimental groups with a voluntary waiting period (VWP) of 40, 120, and 180 d, respectively (G40, n = 135; G120, n = 141; G180, n = 139). Cows of G120 and G180 were reexamined at the end of the VWP. If natural estrus was detected within 46 d after the end of the VWP, an artificial insemination was performed. If no estrus was detected, the respective cows were synchronized by applying the classical Ovsynch protocol. We found no difference in the proportion of cows in which estrus was detected between 40 to 86 DIM or in the days to first estrus between the 3 groups. Estrus detection in this period was lower in cows with body condition score <3 on 90 DIM compared with body condition score ≥3 (61.5 vs. 76.0%) and in cows with high energy-corrected milk production (ECM) on 92 DIM [58.6 vs. 70.1%, for cows with higher and lower than the median (39.9 kg) ECM, respectively]. The proportion of cows that estrus was detected within 46 d after the VWP was greater in G120 (88.9%) and G180 (90.8%) compared with G40 (70.4%). These effects were more apparent in cows with high ECM. The rate of estrus detection and of becoming pregnant in this period was greater for G120 (hazard ratio = 2.2 and 1.6, respectively) and for G180 (hazard ratio = 2.4 and 1.8) compared with G40. Cows in both groups with extended lactation had greater overall first service conception rates (G120 = 48.9%; G180 = 49.6%) and a lower number of services per pregnant cow (G120 = 1.56 ± 0.1; G180 = 1.51 ± 0.1) compared with G40 (36.6%; 1.77 ± 0.1). We observed no difference in pregnancy loss or in the proportion of cows culled up to 305 d of lactation between the 3 groups. The number of Ovsynch protocols per 1,000,000 kg of ECM was reduced by 75% in G180 and by 74% in G120 compared with G40 (5.9 vs. 7.1 vs. 25.1). In conclusion, extending the lactation of dairy cows can improve main reproductive measurements in high-yielding cows.

Normal progesterone profiles during estrus in the bitch: A prospective analysis of 1420 estrous cycles.

Appropriate timing of insemination in the bitch, through monitoring of serial serum progesterone concentrations, has historically been based on the detection of important physiological reproductive events which include the LH surge and ovulation 2-3 days later. Previous hormone profile studies during the estrus period in the bitch, in which determination of these physiological reproductive events has been defined, have been based on low numbers of bitches of similar breeds and body weights. Therefore, the aim of this large-scale prospective study was to define the normal serum progesterone profile during the estrus period in a large number of bitches of various breeds and body weights. In addition, we investigated if the rate of change in progesterone concentrations during the fertile period affected the reproductive performance of bitches after insemination with either fresh or frozen-thawed semen. A total of 1300 individual bitches, representing 84 different breeds, contributed 1420 estrous cycles and 4213 serum progesterone values over the 11-year (2007-2017) study period. The mean (±SD) progesterone concentration at estimated LH0 was 2.7 ± 0.6 ng/ml and at the time of estimated ovulation it was 4.8 ± 0.9 ng/ml and 7.2 ± 1.3 ng/ml (LH+2 and LH+3 respectively). There was no difference in the shape of progesterone profiles for bitches of different body weights. Furthermore, mean progesterone concentrations on each day between LH-6 and LH+7 were not different amongst bitches of different breeds and body weights and there was no effect of mean progesterone concentrations on any day on whelping rate or litter size. However, there was a significant effect of the rate of change in progesterone concentrations on litter size when frozen semen was used. The litter sizes of bitches inseminated with frozen semen with slow progesterone curves were significantly smaller compared to bitches with fast progesterone curves (3.9 ± 1.8 vs 5.6 ± 3.1 pups per litter respectively; P < 0.001). There was no effect of bitch age on the normal progesterone curve, or its rate of change. This is the first report of the normal progesterone profile during estrus in the bitch derived from a large number of serial progesterone measurements obtained from bitches of various breeds and body weights. Importantly, the predictability and reliability of this progesterone profile regardless of breed or body weight gives clinicians the confidence to accurately determine the optimal time for insemination, which is critical to reproductive success, especially when frozen semen is used.

Cortisol and Blood Urea Nitrogen Profiles in Fertile and Repeat-breeder Holstein-friesian Crossbred Cows.

BACKGROUND AND OBJECTIVE: Repeat breeding, in which conception fails after repeated inseminations is a major problem in the dairy industry that leads to economic losses. To examine the role of stress under this condition, this study was conducted to evaluate cortisol and blood urea nitrogen (BUN) levels in fertile and repeat-breeder Holstein-friesian crossbred cows in smallholder farms. MATERIALS AND METHODS: Thirty cows were divided into two groups: Fertile and repeat-breeder cows, with 15 cows per group. Blood samples were collected from the caudal vein at night, 8 h after feeding, once in the oestrus phase during two oestrus cycles and cortisol levels were analyzed by using an enzyme-linked immunosorbent assay with a commercial kit. The BUN was analyzed by the urease and glutamate dehydrogenase method. RESULTS: Significant differences in cortisol and BUN levels were observed between fertile and repeat-breeder cows. Cortisol levels were higher in the repeat-breeder group (6.860±1.427 ng mL-1) compared to those in the fertile group (3.145±1.103 ng mL-1). Furthermore, BUN levels were also higher in repeat-breeder cows (31.45±4.70 mg dL-1) than in fertile cows (27.30±2.69 mg dL-1). CONCLUSION: Cortisol and BUN levels above the normal range may contribute to repeat breeding in Holstein-friesian crossbred cows.

Preovulatory progesterone secretion terminates the duration of reproductive behavior during heat in the bitch.

To evaluate the role of preovulatory progesterone on canine sexual behavior and the course of proestrus and estrus, seven bitches in spontaneous cycles were treated with aglepristone for temporary elimination of progesterone action. Aglepristone was administered at the dose 10 mg/kg b.m., two times 24 hours apart, beginning in early proestrus when progesterone concen- tration was ⟨0.5 ng/ml. Seven untreated bitches served as a control group. Reproductive sexual behavior (standing behavior, display vulva, tail deviation) was evaluated according to behavioral score. Cytologic, clinical and vaginoscopic examinations and progesterone measurements were used for the determination of proestrus and estrus and estimation of ovulation time. Although, a similar pattern and magnitude of sexual behavior were observed in both groups, the duration of a total reproductive behavior was significantly extended (28.71 ± 2.06 vs 17.00 ± 2.45, p ⟨0.05) in experimental group; similarly, the length of cytologic estrus (23.86 ± 3.02 vs 11.14 ± 2.41, p ⟨0.05) was prolonged in treated bitches. In contrast, ovulation rate, duration of proestrus did not differ between the groups (p >0.05). We conclude, that during the canine estrus cycle the preovulatory progesterone terminates the duration of reproductive behavior and cytologic estrus.

Estrus Detection Through Vaginal pH in Saanen Etawah Crossbreed Goats.

BACKGROUND AND OBJECTIVE: In goats, weak estrus behavior is potentially caused by a decline in reproductive efficiency. In the present study, aim was to measure the vaginal pH to determine the accuracy of lust detection in Saanen Etawah crossbreed goats. MATERIALS AND METHODS: Thirteen Saanen Etawah crossbred does (Capra hircus) with body condition score of 2-3 were used. The dose were fed concentrate, provided forage and water ad libitum. The experiment was conducted during the natural estrous cycle. Vaginal smears were collected from each dose every day over a 60 days period to determine the time of each phase of the estrous cycle. The vaginal pH was measured using pH indicator paper; simultaneously, the blood was sampled from the caudal vein into anticoagulant-coated tubes following estrous cycle, covering estrus (E/day-0), metestrus (M/day-3), diestrus (D/day-12) and proestrus (P/day-15). The concentration of plasma estrogen and cortisol was determined by solid phase competitive enzyme-linked immune sorbent assay and the correlation between vaginal pH with each phase of estrous cycle and estrogen level was found. RESULTS: The changes in vaginal pH were confirmed with estrogen and cortisol profile at each estrous phase. The highest vaginal pH and estrogen content were found during estrus, whereas, the lowest vaginal pH and estrogen content was found during diestrus. Furthermore, the cortisol level was significantly different in each phase of the estrous cycle; the highest level of cortisol was observed during proestrus. CONCLUSION: The correlation between vaginal pH and estrous phase indicates that the vaginal pH can be a useful parameter for estrus detection.

Comparing the values of progesterone in the blood of bitches as measured with a chemiluminescence immunoassay and a radioimmunoassay.

A 125 I radioimmunoassay (RIA) has long been used to determine the value of progesterone in serum or plasma of bitches but was discontinued in 2014. A chemiluminescence immunoassay (CLIA) gained prominence since 2003 to determine the value of progesterone in serum of bitches but the assay changed in 2012. This study assessed the agreement between progesterone values obtained with RIA in plasma (progRIA) and with the post-2012 CLIA (progCLIA) in the serum of bitches. ProgCLIA was determined in 110 serum samples from 40 bitches in pro-oestrus or early oestrus and compared to progRIA in plasma samples collected from the same bitches at the same time, where progRIA had a uniform distribution between 0.5 and 25 nmol/L. Two replicate analyses of each serum or plasma sample were simultaneously done in the same assay. For RIA and CLIA, the intra-assay CVs were 5.85% and 6.70% and the interassay CVs 8.45% and 9.16%. For RIA and CLIA the progesterone values obtained with replicate analyses differed by as much as 11%-31% in 25% of samples. On average, the value of progCLIA was 85% of that of progRIA (95% CI 58%-112%, n = 110), with 88% of progCLIAs being lower than the progRIAs. This study shows that RIA and CLIA may yield replicate values that differ by as much as 11%-30% in about a quarter of samples analysed, necessitating replicate analyses if precise values are required. The study provides an equation by which to estimate progCLIA from progRIA.