Early Diestrus Research Articles

Sex- and estrous-related response patterns for alcohol depend critically on the level of compulsion-like challenge

Alcohol use disorder is a substantial social and economic burden. During the last years, the number of women with drinking problems has been increasing, and one main concern is that they are particularly more vulnerable to negative consequences of alcohol. However, little is known about female-specific response patterns for alcohol, and potential underlying differences in brain mechanisms, including for compulsion-like alcohol drinking (when intake persists despite adverse consequences). We used lickometry to assess behavioral microstructure in adult Wistar male and female rats (n = 28–30) during alcohol-only drinking or moderate- or higher-challenge alcohol compulsion (10 or 60 mg/l quinine in alcohol, respectively). Estrous stages were determined and related to drinking levels and patterns of responding to alcohol, as was ovariectomy. Our findings showed that females (where we didn't determine estrus stage) had similar total licks in a session as males, but significantly longer licking bouts under alcohol-only and moderate-challenge, suggesting greater persistence. Further, greater intake under alcohol-only and moderate-challenge was related to faster licking in males, while female consumption was not related to licking speed. Thus, females could have increased persistence without greater vigor, unlike males. However, under higher-challenge, faster licking did predict higher intake in females, similar to males. To better understand female higher-challenge responding, we examined drinking in relation to phases of the estrous cycle. Higher-challenge had longer bouts only in late diestrus. In addition, ovariectomy led to longer bouts only under higher-challenge, suggesting that conditions with reduced hormone levels could increase female persistence for alcohol under higher-challenge. However, ovariectomy also reduced alcohol-only and moderate-challenge drinking but did not reduce bout length. Thus, intake level and response strategy could be regulated somewhat differently by ovarian hormones. Finally, moderate-challenge licking speed was less variable during early diestrus, and we previously showed more stereotyped responding specifically under moderate-challenge in males. By combining behavioral microstructure and sex- and estrus-related changes in drinking patterns, our results suggest that females have greater persistence for alcohol under lower-challenge drinking, while late diestrus and ovariectomy unmasked greater persistence under higher-challenge. Together, our novel insights could help develop more effective and personalized treatments for problematic alcohol use.

Regulation of synthesis of polyamines by progesterone, estradiol, and their receptors in uteri of cyclic ewes†.

Progesterone (P4), estradiol (E2), and expression of their receptors (PGR and ESR1, respectively) by cells of the uterus regulate reproductive performance of mammals through effects on secretion and transport of nutrients into the uterine lumen. This study investigated the effect of changes in P4, E2, PGR, and ESR1 on expression of enzymes for the synthesis and secretion of polyamines. Suffolk ewes (n = 13) were synchronized to estrus (Day 0) and then, on either Day 1 (early metestrus), Day 9 (early diestrus), or Day 14 (late diestrus) of the estrous cycle, maternal blood samples were collected, and ewes were euthanized before obtaining uterine samples and uterine flushings. Endometrial expression of MAT2B and SMS mRNAs increased in late diestrus (P < 0.05). Expression of ODC1 and SMOX mRNAs decreased from early metestrus to early diestrus, and expression of ASL mRNA was lower in late diestrus than in early metestrus (P < 0.05). Immunoreactive PAOX, SAT1, and SMS proteins were localized to uterine luminal, superficial glandular, and glandular epithelia, stromal cells, myometrium, and blood vessels. Concentrations of spermidine and spermine in maternal plasma decreased from early metestrus to early diestrus and decreased further in late diestrus (P < 0.05). The abundances of spermidine and spermine in uterine flushings were less in late diestrus than early metestrus (P < 0.05). These results indicate that synthesis and secretion of polyamines are affected by P4 and E2, as well as the expression of PGR and ESR1 in the endometria of cyclic ewes.

Ovine endometrial estrogen receptor expression is altered following PG-600 administration.

Dysregulation of the estrogen receptor 1 (ESR1) expression during the establishment of pregnancy could contribute to reduce fertility reported in ewes treated with PG-600. The objective of this study was to evaluate the effect of treatment with PG-600 on ESR1 expression in the ovine endometrium during early diestrus. Polypay ewes (n = 24) that had exhibited an oestrous cycle of normal duration (16-18 days) were treated with intravaginal progesterone-releasing device (CIDR) for 9 days. Cloprostenol was administrated two days before CIDR withdrawal. On the day of CIDR withdrawal (day 0), ewes received a 5mL intramuscular injection of PG-600 (400 IU equine chorionic gonadotropin and 200 IU human chorionic gonadotropin) or saline. Blood samples were collected before treatment and on the day of tissue collection to determine serum estradiol-17β and progesterone concentrations using radioimmunoassay. On days 4 and 7, six ewes from each treatment group were anaesthetised and a laparotomy was performed to obtain intercaruncular endometrial samples. Endometrial samples were collected ipsilateral to the ovary bearing the highest number of corpora lutea. An estradiol exchange assay was used to determine ESR1 concentration. Estradiol concentrations did not differ by time or treatment, but progesterone concentrations were significantly higher in the PG-600-treated group on day 7 despite a similar number of corpora lutea. Endometrial ESR1 concentration was significantly reduced on day 7 in the PG-600 group. In conclusion, although progesterone was higher than controls on day 7, ESR1 did not differ from controls suggesting that treatment with PG600 is unlikely to impair reproductive potential.

Structural and Functional Dynamics of the Ovary and Uterus during the Estrous Cycle in Donkeys in the Eastern Caribbean.

Eight non-bred, non-pregnant, regularly cycling Caribbean jennies were examined daily via transrectal ultrasound to define the ovarian and uterine dynamics during four consecutive estrous cycles. Blood samples were collected every other day for progesterone analysis. The mean (±SD) overall inter-ovulatory interval across all donkeys and cycles was 22.93 ± 1.99 days. The maximum follicular diameter was 34.6 ± 2.9 mm. A two-wave pattern was evident in 97% (30/31) of the cycles. The emergence of the future dominant follicle and the largest subordinate follicle of the major primary wave coincided on Day 5.7 ± 3.6 post-ovulation, whereas the secondary wave emerged on Day 19.8 ± 2.9 during estrus of the previous cycle or early diestrus. The secondary wave was often minor (93%, 28/30 cycles). Follicular deviation occurred 8.2 ± 1.4 days before the subsequent ovulation. Luteal volume increased for the first four days after ovulation and reached a maximum volume of 8.5 ± 2.7 mm3 at Day 5.4 ± 0.4, before gradually regressing after Day 15. Serum progesterone concentration increased from Day 1 after ovulation, peaking at 27.0 ± 9.6 ng/mL between 7 and 10 days after ovulation. Progesterone concentration dropped precipitously around Day 15 after ovulation and was below 2 ng/mL around Day 17 ± 2. A day effect (p < 0.0001) was observed for corpus luteum’s volume, progesterone concentration, and uterine tone, but not for endometrial edema (p > 0.05). This study helps to clarify and define normal estrous characteristics of jennies in the Eastern Caribbean.

Analysis of Uterine Blood Flow in Breeding Sows through the Estrus and Early Diestrus, and after Artificial Insemination.

This study aimed to determine uterine blood flow indices by transabdominal Doppler ultrasound in sows (n = 18) under different conditions: (i) sows after estrus detection (day 0, D0); (ii) sows 2 h after artificial insemination (AI), performed 24 h after detection of estrus (day 1, D1); (iii) sows in early diestrus (day 5, D5). Moreover, three different types of seminal doses were used for AI depending on the ejaculate fraction included (F1: doses containing only the rich fraction of the ejaculate; F2: F1 + the transition fraction between rich and poor fractions; F3: F2 and poor fraction). The statistical analysis revealed significant differences in some indices regarding the period of analysis (D0, D1, and D5). Diastolic velocity and mean velocity showed lower values at D5 in comparison with D0 and D1 (p < 0.01). On the other hand, the pulsatility index and the relationship systolic velocity/diastolic velocity indicated higher values at D5 in comparison with D0 and D1 (p < 0.01). No differences were observed regarding the type of seminal dose used in any of the time points analyzed (p > 0.05). Neither insemination per se nor the type of ejaculate fraction used immediately modified the uterine vascularity, but some indices are affected by the stage of the estrus cycle (estrus vs. early diestrus).

Progesterone-dependent and progesterone-independent modulation of luminal epithelial transcription to support pregnancy in cattle.

In cattle, starting 4-5 days after estrus, preimplantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early postestrus, endometrial function is regulated by sex steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. The first objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 days (D4) after estrus. The second objective was to discover luminal transcripts that predict pregnancy outcomes when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome was determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 nonpregnant). Progesterone concentration on D4 was associated positively (n = 182) and negatively (n = 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity, and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n = 22) and negatively (n = 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64%-96% and 44%-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and progesterone-independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.

Dynamics of lipid droplets in the endometrium and fatty acids and oxylipins in the uterine lumen, blood, and milk of lactating cows during diestrus

Our objective was to investigate the lipid content of uterus, blood plasma, and milk at early, mid, and late diestrus. Lactating cows (n = 30) had the estrous cycle and ovulation synchronized by administration of exogenous hormones. Cows were blocked by parity and assigned randomly to receive transcervical uterine flushing and biopsy on d 5 (early diestrus), 10 (mid diestrus) or 15 (late diestrus) of the estrous cycle. Flushing and endometrial biopsy were performed in the uterine horn ipsilateral to the corpus luteum. The recovered flushing was used for analyses of lipid composition by liquid chromatography-tandem mass spectrometry and the biopsy was used for investigation of lipid droplet abundance in endometrial cryosections using a neutral lipid fluorescent dye. In addition, blood and milk samples were collected from all cows on d 5, 10, and 15. All blood samples were used to measure the concentration of progesterone in plasma, and all milk samples were used to determine milk composition. Subsamples of blood plasma and milk were also used to evaluate the composition of fatty acids and oxylipins using the same methodology used for uterine flushing samples. The abundance of lipid droplets in the endometrium increased 1.9-fold from d 5 to 10, and 2-fold from d 10 to 15. Concentration of long-chain fatty acids and oxylipins in uterine flushing were, on average, 2.2 and 2.5 times greater in samples collected on d 15 compared with those collected on d 5 and 10. These differences were not observed in blood and milk, suggesting that accumulation of fatty acids and oxylipins in the uterus is regulated locally. In addition to concentration, the profile of individual fatty acids and oxylipins in uterine lumen changed substantially during diestrus. The main categories with increased abundance at late diestrus were mono- and polyunsaturated fatty acids, and oxylipins derived from arachidonic acid, dihomo-γ-linolenic acid, and docosahexaenoic acid. In conclusion, fatty acids and oxylipins accumulate in the uterine lumen during diestrus and might work as a mechanism to supply these lipids to the developing conceptus at late diestrus, when the onset of elongation occurs and substantial synthesis of biomass and cell signaling by lipid mediators are required.

Partial luteolysis during early diestrus in cattle downregulates VEGFA expression and reduces large luteal cell and corpus luteum sizes and plasma progesterone concentration

Our objectives were to investigate potential changes in the size of steroidogenic large luteal cells (LLC) during partial luteolysis induced by a sub-dose of cloprostenol in early diestrus and to determine transcriptional variations in genes involved in corpus luteum (CL) functions. Cows were subjected to an Ovsynch protocol, with the time of the second GnRH treatment defined as Day 0 (D0). On D6, cows were randomly allocated into three treatments: Control (2 mL saline, im; n = 10), 2XPGF (two doses of 500 μg of cloprostenol, im, 2 h apart; n = 8) or 1/6PGF (single dose of 83.3 μg of cloprostenol, im; n = 10). Before treatments and every 8 h during the 48-h experimental period, blood samples were collected and CL volumes measured. Furthermore, two CL biopsies were obtained at 24 and 40 h post-treatment. The 1/6PGF treatment caused partial luteolysis, characterized by sudden decreases in plasma progesterone (P4) concentrations, luteal volume and LLC size, followed by increases (to pretreatment values) in P4 and luteal volume at 24 and 40 h post-treatment, respectively. However, at the end of the study, P4, luteal volume and LLC size were all significantly smaller than in Control cows. Temporally associated with these phenotypes, there was a lower mRNA abundance of VEGFA at 24 and 40 h, and ABCA1 at 24 h (P < 0.05). In conclusion, a sudden reduction in CL size during partial luteolysis induced by a sub-dose of PGF2α analog on day 6 of the estrous cycle was attributed to a reduction in LLC size, although these changes did not account for the entire phenomenon. In addition to its involvement in reducing CL size, decreased VEGFA mRNA abundance impaired CL development, resulting in a smaller luteal gland and lower plasma P4 concentrations compared to Control cows.

Serum progesterone concentration, volume, and apoptosis of corpora lutea in early, middle and late diestrus in the bitch

The aim was to study number, volume, apoptosis of corpora lutea (CL), and serum P4 concentrations in early, middle, and late diestrus of dogs. Thirty-six bitches were ovari-hysterectomized (OVX): Early Diestrus (Group [G]1; OVX 20 days after end of estrus [DEH]); Mid-diestrus (GII; OVX between 21 and 40 days after DEH), and Late-diestrus (GIII; OVX between 41 and 60 days after DEH). Before OVX a blood sample was collected to quantify P4. After OVX, the number of CL (NCL) was recorded, CL measured using both ultrasonography (US) and caliper (CAL), and the volume (mm3) was calculated. Based on abundances of caspase-3, apoptotic luteal cells were detected. Bitches in early-diestrus had greater P4 concentrations than bitches in mid- and late-diestrus (23.52 ± 3.78 and 10.86 ± 3.88 ng/mL; P < 0.05). The NCL, cumulative USCLV, and CALCLV were similar among diestrus stages (P > 0.30). Bitches with CL (≥5) had twice the serum P4 concentrations as bitches with CL1−2 and CL3−4 (22.71 ± 3.70 and 10.97 ± 4.06 ng/mL; P < 0.05). There were correlations between P4 concentrations with USCLV, CALCLV, and NCL (r = 0.64, r = 0.67, r = 0.44; P < 0.0001). When serum P4 concentrations were adjusted for stages of diestrus, however, there were only correlations during early diestrus. The percentage of apoptotic cells was greater in GIII compared with GI and GII (13.75 ± 2.26 % compared with 4.5 ± 0.68 % and 4.6 ± 1.5 %, respectively; P < 0.05). As days of diestrus increased, number of apoptotic cells increased, and serum P4 concentrations decreased.

Characterization of the endometrial transcriptome in early diestrus influencing pregnancy status in dairy cattle after transfer of in vitro-produced embryos.

Modifications of the endometrial transcriptome at day 7 of the estrus cycle are crucial to maintain gestation after transfer of in vitro-produced (IVP) embryos, although these changes are still largely unknown. The aim of this study was to identify genes, and their related biological mechanisms, important for pregnancy establishment based on the endometrial transcriptome of recipient lactating dairy cows that become pregnant in the subsequent estrus cycle, upon transfer of IVP embryos. Endometrial biopsies were taken from Holstein Friesian cows on day 6-8 of the estrus cycle followed by embryo transfer in the following cycle. Animals were classified retrospectively as pregnant (PR, n = 8) or nonpregnant (non-PR, n = 11) cows, according to pregnancy status at 26-47 days. Extracted mRNAs from endometrial samples were sequenced with an Illumina platform to determine differentially expressed genes (DEG) between the endometrial transcriptome from PR and non-PR cows. There were 111 DEG (false discovery rate < 0.05), which were mainly related to extracellular matrix interaction, histotroph metabolic composition, prostaglandin synthesis, transforming growth factor-β signaling as well as inflammation and leukocyte activation. Comparison of these DEG with DEG identified in two public external data sets confirmed the more fertile endometrial molecular profile of PR cows. In conclusion, this study provides insights into the key early endometrial mechanisms for pregnancy establishment, after IVP embryo transfer in dairy cows.

Body Temperature Fluctuation After Ovariohysterectomy in Dogs in Luteal Phase, Inactive Phase and Pyometra: A Clinical Study of 77 Cases

Progesterone (P4) has a thermogenic effect in the bitch and a decrease in P4 has been associated with a mild transient decrease in body temperature. The aim of this study was to investigate the presence, the extent and the pattern of possible hypothermia after ovariohysterectomy (OHE) in healthy dogs in luteal phase and in bitches with pyometra that underwent therapeutic OHE. Sixty-two healthy dogs and 15 dogs with pyometra that underwent standardized OHE were included. Rectal temperature was measured 1 hour preoperatively (pre-op) and 6, 9, 12, 18, 24, 30, 36, 42, and 48 hours postoperatively (post-op). Serum P4 was measured 30 minutes pre-op and 6 and 24 hours post-op. Healthy dogs were classified in 3 groups based on their initial P4 concentration and in 3 separate groups based on their oestrus cycle stage. The temperature differentiation between the groups of the study was assessed using the repeated measures mixed-design ANOVA analysis. Temperature measured 18-42 hours post-op was significantly decreased compared to pre-op when initial P4 >8 ng/mL but not significantly decreased when P4 <8 ng/mL. Post-op temperature fluctuation in pregnant dogs showed no significant difference compared to dogs in diestrus. Dogs with pyometra had significantly lower temperature 6-48 hours post-op compared to all other groups. Conclusively, mild hypothermia (37.5-37.9°C) may be anticipated 18-42 hours after OHE performed in early diestrus or pregnancy in healthy dogs. More significant hypothermia (37.1-37.5°C) may be anticipated in normally recovering pyometra cases.

Reproductive cycle stage assessment using vaginal cytology evaluation in African lions (Panthera leo)

Vaginal cytology evaluation is an economic, non-invasive technique for indirect monitoring of fluctuations in estrogen concentrations, and thus progression of the estrous cycle. This technique is widely used in domestic dogs for determining timing of artificial insemination. There, however, are only a few reports on the vaginal cytology of non-domestic felids, including lions. This study was conducted, therefore, to describe the vaginal epithelial changes throughout the reproductive cycle of African lions, and to investigate the efficacy of vaginal cytology assessments for predicting reproductive stages. During a 12-month period, reproductive behavioral data and vaginal swabs were collected daily from five lionesses. In total, 541 vaginal smears were evaluated for the proportion of mucosal epithelial cells, neutrophils, bacterial cells, and amount of mucous, cellular debris. One single swab with a large proportion of superficial cells, absence of neutrophils, large number of bacteria, without cellular debris was sufficient for detecting lionesses in estrus. Likewise, one cytology sample with a large proportion of parabasal and intermediate cells, few neutrophils, few bacteria, and large amount of mucous, cellular debris enabled detection of females in advanced diestrus or gestation. To distinguish lionesses in early diestrus from those in an inter-estrous period, at least two consecutive swabs were necessary for satisfactory classification. Overall, evaluation of vaginal cytology samples was an effective technique for differentiation among different stages of the reproductive cycle, confirmation of estrus, and pregnancy diagnosis in lionesses. This technique, therefore, has the potential for application in classifying different stages of the reproductive cycle in other feline species.

Global Transcriptomic Analysis of the Canine corpus luteum (CL) During the First Half of Diestrus and Changes Induced by in vivo Inhibition of Prostaglandin Synthase 2 (PTGS2/COX2).

The canine luteal phase exhibits several peculiarities compared with other species. In early diestrus, the corpus luteum (CL) is, at least in part, independent of gonadotropins, and prostaglandins (PGs) appear to be among its main regulators. This was also observed with the inhibition in vivo of COX2, when also transcriptional capacity, vascularization and immune-related factors were affected. Here, we aimed to further investigate the potential effects of PGs withdrawal on the CL transcriptome by performing deep RNA sequencing (RNA-Seq). Samples from a previous in vivo study were used; bitches were treated for 5, 10, 20, or 30 days after ovulation with firocoxib (Previcox®), a PTGS2/COX2 inhibitor, or a placebo. Analysis of results was performed with SUSHI (framework from FGCZ) and with pathways and functional networks analyzers. Time-dependent effects were also investigated and used for quality control. More highly represented differentially expressed genes (DEGs, P < 0.01, FDR < 0.1) in the early CL (days 5 and 10) referred to proliferation and immune system, while in the mature CL (days 20 and 30) they were related with steroidogenesis. The absence of genes concomitantly affected by the treatment at all time-points suggested stage-dependency in the observed effects. Little effect was observed on days 5 and 10. Day 20 had the highest number of DEGs (n = 1,741), related with increased immune response. On day 30, DEGs found (n = 552) referred to decreased steroidogenesis and vascularization. Our results suggest the presence of strong compensatory effects in the early CL and multidirectional effects toward gonadotropin-dependency of the CL after COX2 inhibition.

Evolution in fixed-time: from synchronization of ovulation to improved fertility

Summary. Synchronization of ovulation and subsequent timed artificial insemination became a popular practice in modern, large scale, dairy and beef commercial operations. Popularity was because it became a serious option to eliminate a laborious and low efficiency activity in the industry: checking cows for heat. Synchronization protocols involve the sequential administration of reproductive hormones to manipulate the estrous cycle to provide a fertile oocyte for insemination at a predictable moment. In the last 20 years, great effort was put forth in protocol development and adjustment; current rates of synchronous ovulations are 80–90%. Moreover, fertility to current protocols is similar to that obtained in cows inseminated after heat detection. The present wave of protocol development proposes to face the challenge of further improving pregnancy success of current protocols. The guiding principle is that, due to the intense metabolism associated with copious milk production, steroid hormone concentrations are insufficient for adequate reproductive function. Thus, new protocols have incorporated strategies to adjust the endocrine milieu and consequently support specific portions of the synchronization process. This paper discusses the positive fertility impact of protocol design that incorporated the following concepts: controlling P4 concentrations during follicle growth to increase oocyte quality; reducing circulating P4 prior to AI to increase proportion of cows pregnant; providing estrogen priming during proestrus to benefit conception and reduce embryo mortality; and optimizing progesterone priming during early diestrus to enhance conceptus survival. It is expected that adoption of these concepts will lead to a continuous increase in fertility of lactating dairy cows.

Supplemental progesterone induces temporal changes in luteal development and endometrial transcription in beef cattle

Long-acting injectable progesterone (iP4) supplementation during early diestrus is a strategy to increase conception rates in cow-calf beef operations. However, iP4 treatment causes early functional and structural regression of the corpus luteum (CL) in a proportion of iP4-treated animals, resulting in pregnancy loss. The hypothesis evaluated was that iP4 accelerates downregulation of sex-steroid receptors (PGR, ESR1, ESR2) during early to mid-diestrus and the upregulation of genes controlling PGF2α secretion (OXTR, PTGS2, AKR1B1) during late diestrus in the endometrium. Ovulations of cyclic, multiparous Nelore (Bos indicus) cows were synchronized, and cows were divided to receive placebo or 300 mg iP4 3 d postovulation (D3). Growth and vascularization of luteal tissue were evaluated by ultrasonography. Blood samples were collected from 3 d postovulation to 3 d after luteolysis, and P4 plasma concentrations were measured by radioimmunoassay. On days 3, 5, 7, 9, 11, 13, and 16 luminal endometrial samples were taken using a cytologic brush. Transcript abundance was measured by qPCR. Structural luteolysis occurred 3 d earlier in cows receiving iP4 compared to the control group. Analyzing only cows that received iP4, those that presented early luteolysis (ie, ≤ D16) showed a decrease in CL area and P4 concentration after D5, compared to the control group. Cows that presented early luteolysis showed a reduced abundance of transcripts on D5 for the ESR2 gene and a greater abundance of transcripts for OXTR and ESR1 on D16, compared to cows that did not present early luteolysis. The iP4-induced early luteolysis can be explained by two nonexclusive possibilities: the activation of uterine mechanisms that trigger early secretion of endometrial PGF pulses and the formation of a subfunctional CL that is prone to early regression.

The effect of hCG, GnRH and PGF2α analogue cloprostenol on the oestrus cycle in jennies

The objectives of this study were twofold. Firstly, the present study was designed to examine susceptibility of the corpus luteum (CL) in early diestrus in jennies; and secondly, to investigate the effect of two commonly used hormonal agents in horses on the induction of ovulation in jennies. The oestrus cycles of eleven jennies were monitored by ultrasound every day. When the dominant follicle reached a diameter of 30 mm, the jennies were treated by intramuscular administration of gonadotropin-releasing hormone agonist lecirelin (GnRH, 50 µg pro toto) in the first oestrus cycle, and human chorionic gonadotropin (hCG, 1500 IU pro toto) intramuscularly in the second oestrus cycle. Prostaglandin F2α analogue cloprostenolum (PGF2α, 0.125 mg pro toto) was administered intramuscularly 2 days after the first ovulation and the interovulatory interval was monitored. This study showed that intramuscular administration of 50 µg of GnRH agonist lecirelin resulted in ovulation within 48 h in 73% of treated jennies. Intramuscular administration 1500 IU of hCG was found to be poorly effective to induce ovulation, with 36% of animals ovulating within 48 h. Intramuscular administration of PGF2α analogue cloprostenol 2 days after ovulation was unsuccessful in attempting to shorten the interovulatory interval in donkeys.

Supplementation with long-acting progesterone in early diestrus in beef cattle: II. Relationships between follicle growth dynamics and luteolysis

The aims were to characterize follicular dynamics in response to supplemental progesterone (P4) and to investigate the relationships between follicular growth and onset of luteolysis in P4-treated cows, submitted or not to artificial insemination (AI). Nonsuckled beef cows detected in estrus were assigned to receive AI or to remain non-AI. Three days after ovulation (ie, D3), AI cows were injected with 150 mg of long-acting P4 (AI + injectable P4 formulation [iP4]; n = 22), and the non-AI cows were assigned to receive 150 mg iP4 (n = 19) or saline (control, n = 19). Between D3 and D21, growth dynamics of the dominant follicles (DFs) was monitored by ultrasonography. Plasma P4 concentrations were measured every other day from D9 to D19. Pregnancy status (ie, P: pregnant and NP: nonpregnant) was examined by ultrasound on D28 to D32. Injectable P4 formulation supplementation decreased average maximum diameter of first-wave DF (DF1). Neither day of emergence of DF2 or DF3 nor the proportion of two- or three-wave cycles were altered by supplemental P4. Daily mean diameter of DF2 and DF3 was also similar between control and iP4 groups. Consistently, daily mean diameter of DF1 in iP4-treated cows was smaller for cows that underwent luteolysis by D15 compared to a later onset. Progesterone concentrations between D9 and D19 decreased earliest in the iP4 group, latest in the control group and was intermediate for the NP-AI + iP4 group. In addition, three-wave cycles presented a delayed decrease on plasma P4 concentrations than two-wave cycles. Further analysis revealed that on two-wave cycles, P4 concentrations on D15 were lowest in the iP4 and NP-AI + iP4 animals compared to the control and P-AI + iP4 groups. Conversely, for three-wave cycles, on D15, P-AI + iP4, NP-AI + iP4, and controls had greater P4 concentrations than the iP4 group. In summary, our data indicate that impairment of first follicular growth was associated with P4-induced shortened luteal lifespan (D14–D15) and that three-wave cycles after AI can be more supportive for pregnancy maintenance in P4-treated cows. We speculate that such conditions play a critical role in the embryonic ability to inhibit iP4-induced early luteolysis reported in part I of this series.

Supplementation with long-acting progesterone in early diestrus in beef cattle: I. effect of artificial insemination on onset of luteolysis

Progesterone (P4) supplementation in early diestrus advances changes in the endometrial transcriptome, stimulating embryonic development. However, it also induces early onset of luteolysis. Occurrence of luteolysis before D16 postmating can be detrimental to fertility. A potential counteracting role of the elongating conceptus on early luteolysis is understood poorly. The aim of the study was to evaluate the effect of artificial insemination (AI; ie, pregnancy) on the temporal dynamics of luteolysis of cows supplemented with P4. Nonsuckled beef cows were inseminated at 12 h after estrus (D0: ovulation) or were not inseminated (no-AI). On D3, the AI cows were assigned to receive a single dose of 150 mg of injectable long-acting P4 via intramuscular injection (AI + iP4; n = 23), and the no-AI cows were assigned to receive iP4 (iP4; n = 21) or saline (control, n = 22). Corpus luteum (CL) development and regression were determined by ultrasonography (US) between D3 and D21. Plasma P4 concentrations were measured on D3 and every other day from D9 to D21. Pregnancy status was determined by US (D28‒D32). iP4 supplementation reduced luteal development (D5–D10) compared to the control group and increased incidence of luteolysis between D14 and D15. On D15, the proportion of cows that underwent luteolysis and plasma P4 concentrations differed between the iP4 group (47.6; 2.10 ± 0.47) and the control group (13.6; 4.40 ± 0.46) and was intermediate in the AI + iP4 group, respectively (26.1%; 3.70 ± 0.45 ng/mL; P < 0.05). The AI effects were due to the pregnant cows (n = 7). Considering nonpregnant cows only, the proportion of early luteolysis in the AI + iP4 group (37.5%) was similar to the iP4 group. Pregnancy was not established in cows having a shortened luteal lifespan. Indeed, interval to luteolysis in the AI + iP4 group (15.50 ± 0.66 d) was similar to the iP4 group (16.38 ± 0.46 d), but less than the control group (17.38 ± 0.40 d; P = 0.05). In conclusion, the effect of AI on extending luteal lifespan occurred exclusively in cows that maintained pregnancy.

49 Collection of Day 7 equine embryos in aluteal cycles in mares

Serial administration of prostaglandin F2α (PGF2) in early diestrus has been recently shown to prevent luteal function in mares and is associated with delayed embryo development in induced aluteal cycles (progesterone deprived environment; &amp;lt;1.0ng mL−1; Leisinger et al. 2018 Theriogenology 105, 178-183). We hypothesised that embryos collected on Day 7 during induced aluteal cycles would be developmentally delayed compared with those collected from control cycles (progesterone deprived environment but supplemented with exogenous progestogen). Mares were monitored until a preovulatory follicle=35mm and the presence of uterine oedema were detected by ultrasonography. Mares were treated IV once with 2000IU of human chorionic gonadotropin and artificially inseminated every other day with total motile spermatozoa from one stallion of known fertility until ovulation. Mares were examined twice daily to determine the occurrence of ovulation. After ovulation, mares were randomly assigned to the control group (serial PGF2 treatment+long-acting altrenogest) or AL group (serial PGF2 treatment only). Using a protocol to induce aluteal cycles (Leisinger et al. 2018 Theriogenology 105, 178-183), mares in the AL group (n=7) were treated twice daily with 10mg of PGF2 (Lutalyse, dinoprost tromethamine, Zoetis, Florham Park, NJ, USA) IM on Days 0, 1, 2, and then once daily on Days 3 and 4. Mares in the control group (n=4) were treated with serial PGF2 treatment as mentioned before, and treated with a single injection of 225mg altrenogest (BioRelease Altrenogest LA 150; BET Pharm, Lexington, KY, USA) IM at the time of ovulation. On Day 7 post-ovulation, embryo collection was performed by uterine flushing using lactated Ringer’s solution. The developmental stage of embryos, diameter, and quality were determined using a stereomicroscope and photographed. Embryos were washed 3 times in commercial embryo holding medium (EmCare™ Holding Solution, ICP Bio, Spring Valley, WI) and stained with 1µg mL−1 of 4’,6-diamidino-2-phenylindole at 38.5°C for 15min to determine the number of nonviable cells. Data were analysed using t-test or Mann-Whitney U test where appropriate. Statistical significance was set at P=0.05. Data are reported as mean±s.e.M. Overall, the developmental stage of control embryos differed from AL embryos (P&amp;lt;0.03). In the control group, all embryos (n=4) collected were classified as expanded blastocysts. In contrast, embryos (n=7) collected from the AL group were all early blastocysts. Furthermore, the mean embryo diameter differed in control v. AL groups, 401±8mm v. 166±2mm, respectively (P&amp;lt;0.001). The quality grades of control embryos (1.3±0.3) were not different from the AL group (1.3±0.2; P&amp;gt;0.05). All embryos of both groups had less than 10% of blastomeres stained with 4’,6-diamidino-2-phenylindole. This study showed that morphologically normal equine embryos of acceptable quality can be collected on Day 7 in aluteal cycles.

Using in-line milk progesterone data to characterize parameters of luteal activity and their association with fertility in Holstein cows

Our objectives were to characterize parameters of luteal activity based on milk progesterone concentration (P4c) data from before and after artificial insemination (AI) and to evaluate their potential association with fertility in Holstein cows. Records of AI events (n = 4,353) and of milk P4c (n = 158,961) obtained through an in-line milk analysis system (Herd Navigator, DeLaval International, Tumba, Sweden) from 1,891 lactations of 1,423 Holstein cows were evaluated. Milk P4c (ng/mL) were measured every 2.2 ± 1.9 d (mean ± standard deviation) between 23.6 ± 7.3 and 185.3 ± 56.7 d in milk. Variations in milk P4c of consecutive records were used to determine onset of luteal phase (increase in P4c from <5.0 to ≥5.0 ng/mL), luteal phase length (period, in days, of P4c ≥5.0 ng/mL), cessation of luteal phase (decline from ≥5.0 to <5.0 ng/mL, designated as P4c-decline), and pregnancy (AI followed by a luteal phase that remained uninterrupted until 50 d post-AI). The length of the luteal phase preceding AI, the highest P4c (P4c peak) during the luteal phase preceding AI, the lowest P4c preceding AI (P4c pre-AI) that followed a P4c-decline, and the interval between P4c-decline and AI were evaluated, as well as the interval between AI and onset of luteal phase, and P4c at early diestrus (4.5 ± 0.6 d post-AI), mid diestrus (10.0 ± 0.6 d post-AI), and late diestrus (14.1 ± 0.6 d post-AI). Data were analyzed using logistic regressions, and comparisons made based on quartiles and cut-points established by receiver operating characteristic curve analysis. Overall probability of pregnancy was 32.0%. Parameters associated with reduced probability of pregnancy (represented as percentage points decrease in the probability of pregnancy) were (1) luteal phase length >14.4 d (7.6% decrease), (2) P4c peak ≤24.7 ng/mL (4.5% decrease), (3) P4c pre-AI >0.5 ng/mL (5.5% decrease), (4) interval between P4c-decline and AI of >1.6 d (4.0% decrease), (5) interval between AI and onset of luteal phase of <7 or >11 d (9.3 and 12.1% decrease, respectively), and (6) P4c at early diestrus ≤0.7 or >3.5 ng/mL (15.2 and 6.7% decrease, respectively), (7) P4c at mid diestrus ≤12.4 ng/mL (12.5% decrease), and (8) P4c at late diestrus ≤22.7 ng/mL (9.7% decrease). The parameters of luteal activity associated with reduced probability of pregnancy established here could be used as benchmarks while developing recommendations to improve reproductive performance in herds using in-line milk progesterone monitoring.