Ovulation In Mares Research Articles

Pathohistological Findings after Bilateral Ovariectomy in Mares with Behavioral Problems

Behavioral problems in reproductively healthy mares are a challenging issue that is successfully treated with bilateral ovariectomy (BO). This laparoscopic procedure represents an alternative to conservative treatment for mares not intended for breeding and results in high owner satisfaction regarding behavioral improvement. However, a pathohistological explanation to justify surgical ovarian removal regarding animal welfare is lacking. Therefore, the objective of this study was to pathohistologically evaluate bilaterally removed, clinically unremarkable ovaries of mares with behavioral problems (bOE, n = 20) and to compare them with pathohistologically confirmed granulosa cell tumors of mares with neoplastic ovaries (GCT-uOE, n = 10). A complete data set including preliminary presentation, clinical examination, and serum anti-Müllerian hormone (AMH) and testosterone was further analyzed in both groups. Both hormones were significantly higher in GCT-uOE compared with bOE. Immunohistochemical expression of Ki-67, AMH, aromatase, epidermal growth factor receptor, calretinin, and epithelial cadherin in granulosa cells of large follicular structures in bOE did not differ from neoplastic granulosa cells in GCT-uOE. Ultrasonographically nondetectable early neoplastic changes were pathohistologically evaluated in 15% of mares and anovulatory-like follicles in 30% of mares in bOE and might be one explanation for the high success rate of BO in 85% of bOE in this study.

The use of surfagon for the induction of ovulation of mares in ART cycles

In order to predict the timing of ovulation in mares, to obtain mature oocytes and follicular fluid in ART cycles, the domestic analogue of GnRH surfagon was used in two doses (40 and 50 mcg) against the background of comparison with the effectiveness of the previously used hCG drug. Surfagon injections were carried out intramuscularly when the leading follicle reached a diameter of 35 mm or more, and there was maximum or decreased edema of the uterus. An analysis of the duration of the intervals between intramuscular injection of 40 µg and 50 µg of the drug and ovulation showed that 72.4 and 35% of ovulations occur, respectively, between 37 and 48 hours after injection. The result of using 40 µg of surfagon is similar to the results of using hCG - 72 and 74% of ovulations in this period, respectively. Therefore, the drug “Surfagon” at a dose of 40 µg is most suitable for use in ART cycles. Only 3.4% of ovulations after the use of 40 µg of surfagon occur in the interval up to 24 hours after injection. Therefore, its use allows planning the collection of follicular fluid and oocyte from the preovulatory follicle using the OPU method after 24 hours. The domestic drug Surfagon (lyuleberin acetate, CJSC Mosagrogen, Moscow) is effective for the induction of ovulation in mares and can serve as an alternative to the use of HCG and imported GnRH analogues both in normal reproduction practice and in planning OPU procedures for the purpose of follicular fluid intake and oocyte extraction.

Pregnancy rates according to follicle diameter and uterus edema in different age groups in two consecutive ovulations in Arabian mares

The study aimed to evaluate the pregnancy rates of mares in different age groups based on follicle diameter and uterine edema during two consecutive ovulations in three breeding seasons. Thirty Arabian mares were divided into two groups: below and equal to 10 years (Group A=54) and above 10 years old (Group B=36). Throughout the breeding season, the diameters of ovulatory follicles and uterine edema were recorded. After the first ovulation, when the follicle diameters of all mares in both Group A and Group B were analyzed independently of age, and compared between pregnant and non-pregnant mares, no differences were found (P>0.05). The differences between the agerelated groups were insignificant (P>0.05). Follicle diameters between pregnant and non-pregnant mares at the second ovulation were significantly different (P0.05). Differences between uterine edema values determined in mares that were pregnant and nonpregnant in two consecutive ovulations were found to be insignificant in terms of pregnancy (P>0.05). As a result, although preovulatory diameter is not a reliable marker for pregnancy detection, it remains an important parameter for determining ovulation and artificial insemination timing. Uterine edema is only useful for determining the timing of insemination and is not associated with pregnancy. Moreover, since similar pregnancy rates were observed among different age groups, age alone is not a sufficient parameter for assessing reproductive efficiency in mares.

Comprehensive analysis of the whole-transcriptome landscape of the ovarian cortex from Mongolian horses that reproduce seasonally

The reproductive cycle of equines tends to be seasonal and is influenced by factors such as light and temperature. The process and methods of regulating the mare oestrous cycle in the anestrus period are still immature. The effects of noncoding RNAs and mRNAs on the oestrous cycle have aroused much interest, but corresponding analyses of seasonal mare ovaries have not been reported. Here, we report a whole transcriptome analysis of the Mongolian horse ovarian cortex collected in anestrus and diestrus periods. In total, 1081 mRNAs, 205 lncRNAs, 54 circRNAs, and 13 miRNAs were upregulated in winter anestrus ovarian cortex (WAO), and 1261 mRNAs, 90 lncRNAs, 29 circRNAs, and 40 miRNAs were upregulated in summer diestrus ovarian cortex (SDO). The GO and KEGG enrichment analysis of differentially expressed mRNAs and target genes of differentially expressed lncRNAs, circRNAs, and miRNAs revealed some key functions and pathways that may be related to follicle and oocyte development. We found that estrogen-related pathways were enriched in different RNAs. Our data were used to generate miRNA, circRNA, lncRNA, and mRNA databases from the Mongolian horse ovary and differential expression profiles between WAO and SDO; these results provide clues for exploring methods of estrus regulation in mares during the anestrus period.

Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification.

The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares' uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development. These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

Increase of Body Temperature Immediately After Ovulation in Mares.

To successfully inseminate mares, precise detection of ovulation time is crucial, especially when using frozen-thawed semen. Monitoring body temperature, as has been described in women, could be a noninvasive way to detect ovulation. The objective of this study was to investigate the relationship between the time of ovulation and the variation of body temperature in mares based on automatic continuous measurements during estrus. The experimental group included 21mares for 70analyzed estrous cycles. When the mares showed estrous behavior, they were administered intramuscular deslorelin acetate (2.25mg) in the evening. At the same time, monitoring of body temperature using a sensor device fixed at the left lateral thorax was started and continued for over 60hours. In 2-hour intervals, transrectal ultrasonography was performed to detect ovulation. Estimated body temperature in the 6 hours following ovulation detection was on average 0.06°C +/- 0.05°C (mean+/-SD) significantly higher when compared with body temperature at the same time on the preceding day (P=.01). In addition, a significant effect of PGF2α administration for estrus induction on the body temperature was found, being significantly higher until 6hours before ovulation compared to that of uninduced cycles (P=.005). In conclusion, changes in body temperature during estrus in mares were related to ovulation. The increase in body temperature immediately after ovulation might be used in the future to establish automatized and noninvasive systems to detect ovulation. However, the identified temperature rise is relatively small on average and hardly identifiable in the individual mares.

Comparison of the efficacy of altrenogest versus transdermal progestin patches on estrus synchronization and ovulation in mares.

The adhesive progestin patch was investigated for estrus synchronization in mares because this method is convenient and safe in other species. This study aimed to compare the efficacy of a transdermal progestin patch versus oral altrenogest on estrus synchronization, preovulatory follicle development, and ovulation. Twenty-four broodmares were randomly divided into two groups. In Group 1, mares (n = 12) were fed 0.044 mg/kg altrenogest daily for 14 days. Group 2 (n = 12) mares were treated with adhesive transdermal progestin patches for 14 days. Mares were observed for estrus daily, from day 3 until day 7, after hormone withdrawal. Follicular development was determined by ultrasonography. The preovulatory follicle size and ovulation time after hormone treatments and ovulation rate were determined for both groups. Plasma progesterone levels were measured during the experimental periods and 2 days after hormone termination in both groups. The results revealed that the transdermal progestin patch efficiently controlled follicular growth and estrus synchronization in mares. The percentage of mares exhibiting estrus was similar in the altrenogest (100.00%) and transdermal patch (91.67%) groups. Ovulation rates were equivalent with either altrenogest or progestin patch protocols (91.67% [11/12] vs. 83.33% [10/12]). In addition, the preovulatory follicle size was similar in mares treated with altrenogest and progestin patches. This study revealed that the transdermal progestin patch provides effective estrus synchronization and ovulation, similar to altrenogest treatment. However, the transdermal patch was more convenient with a shorter predictable ovulation time after estrus synchronization and should be considered as an alternative method for mares.

Efficacy of buserelin for induction of ovulation in mares

The rational and controlled management of equine reproduction in commercial programs promotes the development of drugs that help to regulate certain physiological processes in mares, such as luteolysis or ovulation. Human Chorionic Gonadotropin is used predominantly in commercial and experimental programs showing optimal ovulatory results in multiple studies. However, a lower inducing effect has been reported in old mares, which are highly represented in the commercial breeding population. Some studies with human formulations of buserelin, one of the oldest GnRH synthetic analogs, have demonstrated similar efficacy to other analogs in mares (Newcombe and Cuervo-Arango. Journal of Equine Veterinary Science. 2016; 41, 57). In Argentina, until now, there are no specific buserelin formulations for mares. The aim of this clinical prospective study was to compare the ovulation induction efficacy of two doses of a new buserelin formulation in mares of different age groups. The study was carried out in a commercial breeding program in the subtropical north of Argentina using healthy Quarter Horse and Criollo mixed breed mares randomly assigned to treatment groups. When uterine edema, a relaxed cervix and 35 mm follicles were detected, mares were induced once, with a single intravenous (IV) injection of 0.2mg of buserelin (Busereleq,Laboratorio CIMOL, Argentina) for group 1 (G1, n=77) and 0.4mg for group 2 (G2, n=54). The response was considered positive when absence of the dominant follicle and presence in the same ovary of a corpus luteum were detected by ultrasound, 48 hours after treatment. In both groups, mares were classified according to age in <10; 11-15; >15 years old (yo). The data were analyzed by the chi square test for categorized data using Infostat Software. The follicular size, degree of edema and age were compared in the different groups at the time of treatment by means of a non-parametric Kruskal-Wallis analysis of variance. The results showed that 87.01% of the G1 (0,2 mg) and 70.37% of G2 (0.4mg) mares responded positively (p<0.05). No significant differences were found between follicular size, degree of edema and age of the mares at the time of induction among treated groups (p >0.05). Efficacy of the different treatments by age group in G1 and G2 was 77% and 68% in <10 y.o; 96% and 71% in 10 -15 y.o; 90% and 73% in >15 y.o respectively. A significant difference among treated groups was detected in middle age (10-15 years) with more efficacy in G1 (p=0,017). Our results suggest that a low dose buserelin (0.2mg IV) can be used to induce ovulation with acceptable clinical efficacy, even in older mares.

Impact of ovulation inducers on ovarian artery in mares: pulsatility and resistivity indexes

A failure of vascular development in reproductive organs may be the cause of several dysfunctions during the estrous cycle and gestation. Therefore, it is important to evaluate the in vivo influences of ovarian vaso- irrigation on ovulation. The ovarian follicles need an adequate blood supply for oxygen, nutrients, and hormones, besides eliminating CO2 and other metabolites (Bruno et al. Animal Reproduction. 2009; 6, 2:371-379). Transrectal ultrasonography (US) with color, power, and spectral Doppler US has been used to study blood flow and ovarian perfusion in mares (Acosta et al. Biology of Reproduction. 2004b; 71, 2: 502-507). Studies have shown a daily increase in vascularization of the wall of the dominant follicle as it approaches the day of ovulation (Gastal et al. Animal Reproduction Science. 2007; 102, 3-4: 314-321); However, a few hours before ovulation, an abrupt decrease in blood perfusion of the follicular wall has been detected (Ginther et al. Journal of Equine Veterinary Science. 2007a; 27, 1: 20-27). The drugs most used as ovulation inducers in horses are human chorionic gonadotropin (hCG) and analogues of gonadotropin-releasing hormone (GnRH), such as deslorelin (Chavatte & Palmer Equine Veterinary Education. 1998; 10, 26-30). Using different ovulation inducers, this study aimed to evaluate changes in ovarian arteryblood flow after induction of ovulation in mares with follicles ≥ 35mm diameter. Twenty-eight estrous cycles were followed, randomly and equally distributed: GI: control mares without treatment; GII: 1000 IU IV of hCG; GIII: 750µl IV of deslorelin; GIV: 250µg IM of hysterelin. The ovarian artery pulsatility and resistivity indexes (PI and RI) were measured. Thereafter, estrous cycles were followed every 12 hours from time 0 (induction) until ovulation using B-mode US and spectral mode Doppler US of ovarian arteries. Statistical analysis was performed with ANOVA, a p-value of <0.05 was considered significant. No statistical difference was found in PI between groups (p=0.1297) of at the different evaluation times. When the times of analysis were compared separately between the groups, no statistical difference was found between 0h (p= 0.7092), 12h (p= 0.5148), 24h (p=0.2642), and 36h (p=0.582). Similarly, the RI did not differ between the groups analyzed (p= 0.1397), regardless of the time of evaluation, nor between the timepoints analyzed separately between 0h (p= 0.5049), 12h (p= 0.7334), 24h (p=0.3589) and 36h (p=0.1621). It can be concluded that although an increase in vascularization of the wall of the dominant follicle occurred, the PI and RI of the ovarian artery were not influenced by any ovulation inducer of the pre-ovulatory follicles.

HCG versus histrelin acetate: ovulation inducing agents in mares (Equus caballus) in a commercial breeding program in Uruguay

Hormonal control of ovulation timing is an effective tool to optimize reproductive management and reduce costs in equine breeding programs. Few studies have evaluated the efficacy of GnRH analog histrelin as an ovulation inducer agent. This study aimed to compare the efficacy of human chorionic gonadotrophin (hCG) and histrelin as ovulation inducers in young, adult, and old mares during spring and summer. Reproductive records of 321 estrous cycles of 125 mares, mean age 9.8±0.37 years, range 1.5–24 years, between 360-600 kg body weight, and body condition ≥3 (1/5), were evaluated during spring (151 cycles) and summer (170 cycles). Mares were kept in the field and fed alfalfa hay, balanced concentrate feed and ad-libitum water. The ovaries, uterus, and cervix underwent transrectal examination and ultrasonography daily. When only one dominant follicle of 35 mm and endometrial edema compatible with estrus were reached, ovulation was induced. Each cycle was monitored every two to six hours until ovulation, 24h after induction. Cycles were randomly allotted in two groups; G1 (n=106 cycles): 1500 IU hCG (IV); G2 (n=215 cycles): 250 μg histrelin acetate (IM). A total of 119 cycles were from young (<8 years), 93 from adults (8-15 years), and 109 from old mares (>15 years). Cycles of mares ovulating within 24-48hafter treatment were considered to have responded to the inducer, and those ovulating before 24h and after 48h were excluded from the experiment. Inducer agents, season (spring or summer), and mare age were independent factors, and ovulation between 24-48h was considered the dependent factor. Data were analyzed through Fisher's exact test. The significance level was defined as P<0.05. From the 321 induced cycles, 254 (79.1%) ovulated within 24-48h, 20 (6.2%) before 24h, and 47 (14.6%) after 48h. There were no differences (P=0.2414) in the average response time between G1 (37.2h±3,5) and G2 (39.3h±4,5). The ovulatory response between G1 and G2 did not differ in spring (84.1% versus 90.7%, P=0.2627) and summer (79.2% versus 85.4%, P=0.3534). Independent of the inducer, the season did not influence the ovulation response in the different mare ages (spring P=0.1489; summer P=0.2992). No differences (P=0.896) were found between drugs in young and adult mares. However, old mares had a higher percentage of ovulatory response in spring and summer to histrelin (n=76) than to hCG (n=24) (89.5% versus 45.8%, P<0.0001). In conclusion, histrelin is more effective and accurate in inducing ovulation in old mares (>15 years) than hCG throughout the breeding season.

Equine endometrial Th1 and Th2 evaluation during early pregnancy

As in all mammals, continuous feto-maternal dialogue is essential for successful pregnancy in mares. Nevertheless, several features of pregnancy in the mare are unusual and differ markedly from equivalent events in other species. The nature of the signal by which the equine embryo biochemically indicates its presence in the mare´s uterus for pregnancy maintenance remains a mystery to the present day, but the equine conceptus must begin to release this factor resulting in maternal recognition of pregnancy around day 10 to day 13 after ovulation. In humans and some animal species, the Th1/Th2 balance is essential for immunological tolerance in pregnancy. This immunohistochemical study aimed to assess the T-bet (Th1) and GATA3 (Th2) lymphocyte populations in the endometrium on days 7, 10, and 13 after ovulation in cyclic and pregnant mares. Mares were randomly assigned to the day of sample collection in both groups. In the first cycle, endometrial biopsies from 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10), and 13 (n = 10) post-ovulation (Cyclic group). In the second cycle, the same mares were bred to a fertile stallion, and endometrial samples were collected on days 7, 10, and 13 post ovulation. Immediately after biopsies, uteri were flushed, and those with an embryo recovered were allotted to the Pregnant group. Of the 30 mares flushed, embryos were recovered from6 mares on day 7, 6 on day 10, and 6 on day 13. Samples from mares without embryo recovery were excluded from both groups. Immunohistochemistry was used to detect Th1 (T-bet PA5-28881, Invitrogen) or Th2 (GATA3 L50-823, Thermo Fisher) proteins in the endometrium. Only stained lymphocytes from the stratum compactum (SC) were counted. Five fields were analyzed at 400x magnification. A two-way ANOVA was performed to evaluate the absolute number of T-bet/GATA3 cells and their ratio as a dependent factor and experimental groups, day, and their interactions as independent variables. T-bet-stained cells decreased from days 7 to 13 in both the Cyclic (day 7=73 vs 13=48) and Pregnant groups (day 7=83 vs 13=39) (P=0.0179); no difference was observed between groups. GATA3 stained cells remained constant over the days, with more stained cells in the Pregnant (day 7=37; 10=35; 13=34) than in the Cyclic group (day 7=23; 10=12; 13=12) (P=0.0001). The Th1/Th2 ratio did not show a difference (P>0.2222). Th2 cells are recruited into the SC of the endometrium of the pregnant mare. These cells could be secreting interleukins and establishing an anti-inflammatory microenvironment in early pregnancy. In conclusion, the presence of Th2 cells in pregnant mares could be important for embryonic development and maternal recognition of pregnancy regarding immunological tolerance.

Pathohistological findings in bilateral removed ovaries of mares with behavioral problems

Unwanted behavior is a common presenting problem in mares which often challenges veterinarians. Behavioral problems associated with cyclicity in absence of neoplastic ovarian pathology are often treated with oestrus suppression (Aurich and Kaps, Reprod.Dom.Anim. 2022;00:1-9). However, most options are cost-intensive, might have severe side effects or reversibility is not clearly determined yet. Therefore, bilateral ovariectomy (bOE) is often chosen due to the benefited surgical procedure. Studies evaluating effects of bOE on unwanted behavior in mares from the owner´s perspective revealed an improvement (Melgaard et al., Animals 2020;10:1210). However, there is a lack of histopathological explanation for this phenomenon. Therefore, the aim of the present study was to evaluate bilateral removed ovaries histopathologically in mares with behavioral problems. We hypothesized, that there are non-neoplastic changes in ovaries histologically detectable, which could be responsible for unwanted behavior in mares. In a retrospective case series, ovaries of 20 mares after bOE (OE) via standing laparoscopy using routine procedures (Röcken et al., Vet. Surge. 2011;40:1009-1014) were compared with granulosa cell tumors (GCT, n=10). Samples from removed ovaries were fixed in formaldehyde for further histological evaluation. One additional representative sample of each ovary was sent to a commercial laboratory for histopathological diagnosis of GCT by a Board-certified pathologist.Immunohistochemical evaluation was carried out using Kiel 67 (ki67), Aromatase (AR) and Anti-Muellerian Hormone (AMH) as diagnostic marker. Intensity of staining for ki67, AR and AMH was classified as -, +, ++, and +++ for no, low (<40%), moderate (40-70%) and high (70-100%) expression, respectively. Immunolabeling for ki67, AR and AMH was most intense in granulosa cells of antral and early atretic follicles in OE. In GCT, immunolabeling for AR and AMH was additionally highly intense in Leydig-like cells. In OE, ovaries of 3 mares initially categorized as GCT could not be confirmed and were therefore regarded as early stages due to upregulated ki67 in presence of a corpus luteum. Measurements of AMH in serum performed before surgery were above the reference value in all mares of GCT and in 6/20 (30%) in OE. In GCT, 8/10 (80%) and in OE, 16/20 (80%) mares showed behavioral abnormalities before surgery, which were not correlated with increased AMH in serum. Serum AMH values were not correlated with high expression of AMH in 9 mares, where granulosa cells of antral follicles could be evaluated. All, but one mare, benefit from bOE regarding behavior as reported from the owner. In the present study, immunostaining with ki67, AR or AMH showed no clear difference between OE and GCT. Further investigations regarding different immunohistochemical staining are warranted to find marker for non- or early neoplastic changes related to improvement of behavior in mares after bOE.

Rationale and efficacy of sexual desire stimulation in mares under anaphrodisia

Data from the literature regarding the objectivity of clinical and hormonal methods of diagnosing disorders of the gonads are contradictory, because they do not always accurately reflect the state of the course of folliculo-luteogenesis. At the same time, the relationship between clinical and endocrine indicators is still insufficiently clarified, which does not allow for the development of a justified use of drugs that accelerate the manifestation of sexual hunting in mares. In this regard, our further studies were aimed at studying the dependence of hormonal indicators on the size, shape and consistency of the gonads and determining the effectiveness of stimulation of sexual hunting with prostaglandin F 2α drugs. Before conducting the experiment, it was taken into account that exogenous prostaglandin F-2 alpha has both luteolytic properties - causes lysis of the corpus luteum, and stimulating properties - affects the release of gonadotropin hormones (FSH, LH) by the hypothalamus and pituitary gland, which leads to an acceleration of the manifestation of phenomena arousal stage of the sexual cycle. From the data presented in the article, it can be seen that after the use of estrophan sexual desire was found in a larger number of animals than when bioestrophan was administered and in the control. It was observed that the effectiveness of bioestrophan compared to estrophan was lower according to some parameters: 29% of animals showed less desire for sex, its duration was 1.5 days longer (8.2±1.6 vs. 6.7±0.6), and fertilization is 19% lower. The reaction of the animals to the administration of the drugs in terms of the period from administration to the manifestation of potential libido (desire with clear signs) was almost the same (7.0±1.8 and 7.3±1.2 days). The effectiveness of prostaglandin F-2 alpha preparations, applied to mares with anaphrodisiacs to stimulate the onset of the arousal stage of the sexual cycle, depends on the value of IPRYA (the value of the integral indicator of the size of mare's ovaries), which characterizes the activity of the gonads and their refractoriness to exogenous prostaglandins. With a large value of IPRYA - 14–18 cm, compared to the control, the introduction of estrophan and bioestrophan increased the number of mares that showed sexual desire by 47.5 and 27%, accelerated the onset of sexual hunting by 9 and 8 days, contributed to an increase in fertilization by 43 and 30%. Based on the above, the PI of mare's ovaries can be used to clarify the diagnosis - hypofunction and persistence of the corpus luteum of the gonads. Key words: sexual cycle, sexual desire, exogenous prostaglandin F-2 alpha, anaphrodisia, infertility, mares.

Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production

Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 μg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 μg/ml (H60, n = 196), 164 μg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32–76%; median, min-max) in comparison to H60 and H164 (24%,11–33% and 13%, 8–44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P < 0.01). Blastocyst rates (blastocyst/cleaved oocytes) and total cell number were not different among the groups (control 29%, H60 25%, and H164 24%). In a preliminary study, equine oocytes (n = 28) were exposed to PB in vitro for 6 h followed by intracytoplasmic sperm injection (ICSI) and in vitro embryo production. Exposed oocytes showed a numerically lower maturation rate (43% Vs 52%; P > 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained.

Ovariectomy in a mare with granulosa cell tumor: Case report

Granulosa cell tumor is the most common type of neoplasm that affects the ovaries of mares. It is usually benign and unilateral, while the contralateral ovary is usually atrophied and without follicular activity. The diagnostic suspicion is based on clinical signs and behavioral changes, such as anestrus and increased libido; however, it is confirmed by hormonal, ultrasound and histopathological examinations. The aim of this study is to report a case of granulosa cell tumor in an 18-year-old female horse, submitted to an ovariectomy procedure and pre- and postoperative care. Surgical intervention proved effective, not only preventing damage that could be caused by the tumor, but also restoring follicular activity in the contralateral ovary.

The effect of dexamethasone and flunixin-meglumine on ovulation, endometrial oedema, and inter-ovulatory interval length in the mare

The use of flunixin-meglumine (a potent non-steroidal anti-inflammatory drug) during the critical period of intrafollicular prostaglandin production before ovulation (24 and 36 h after hCG treatment) results in a high rate of ovulatory failure and formation of haemorrhagic anovulatory follicles (HAF) in the mare. Dexamethasone is commonly used to prevent persistent mating-induced endometritis in susceptible mares, but the effect on ovulation blockage within the pre-ovulatory critical window of intrafollicular prostaglandins production following hCG administration has not been determined. Six mares were followed during four consecutive cycles in a crossover design; once in oestrus with a follicle of >32 mm in diameter, mares were treated with hCG (Hour 0) and assigned to one of 4 groups randomly: 1) FM, mares received 1.7 mg/kg flunixin-meglumine at Hour 24 and 36; 2) CON, mares received no further treatment. 3) DEX1, mares received 0.1 mg/kg dexamethasone at Hour 24, and 4) DEX2, mares received 0.1 mg/kg dexamethasone at Hour 24 and 36. For all groups, ovulation and HAF rates, endometrial oedema profiles and the inter-ovulatory intervals (IOI) were determined and compared statistically. All CON and DEX mares ovulated normally and did not form any HAF. On the contrary, FM mares developed a HAF in 83% of cycles (P < 0.01). The endometrial oedema score was lower following DEX administration than FM (P < 0.05). The mean IOI was longer (P < 0.05) in DEX1 and DEX2 groups (26.5 and 26 days, respectively) than in CON and FM groups (21.5 and 22 days, respectively). In conclusion, dexamethasone treatment given either once or twice during the critical window of hCG-induced ovulation did not block or delay ovulation, but had a similar ovulation rate than untreated control mares. However, the inter-ovulatory intervals of dexamethasone treated mares was longer than control and FM treated mares. Finally, dexamethasone treatment was more effective in reducing endometrial oedema than FM.

Field observation on the use of PRID®Delta to induce estrus and ovulation in anestrous mares

To advance the time of the first estrus with ovulation, we administered intravaginally a progesterone-releasing devices PRID® Delta to 42 acyclic Arab-Barb mares for 11 days at different seasons: winter (n = 10), spring (n = 14), summer (n = 9) and autumn (n = 9). Twenty-seven of 39 mares that kept their coils responded to the PRID® Delta treatment with estrus and ovulation during the 42-day observation period, in winter (4/10, 40%) mares, in spring (9/13, 69%), in summer (7/7, 100%) and autumn (7/9, 78%) mares (P &lt;0,05). At Day 2 of PRID® Delta treatment, mean initial of progesterone concentrations was significantly increased in all mares in winter (7.20 ± 0.49 ng mL-1), spring (7.30 ± 0.64 ng mL-1), summer (7.5 ± 0.58 ng mL-1 ) and autumn ( 7.60 ± 0.71 ng mL-1) (P &lt;0,05). Total concentration of progesterone (area under curve (AUC)) during the treatment period revealed highest values in spring (67.95 ± 2.40 ng mL-1h-1) followed by autumn (65.20 ± 1.37 ng mL-1 h-1), winter (54.19 ± 7.00 ng mL-1 h-1) and summer 52.23 ± 3.32 ng mL-1 h-1; P &lt; 0.05). In conclusion, administration of the intravaginal device releasing progesterone PRID® Delta was able to induce estrus and ovulation in mares at different times of the year. However, the efficacy of the treatment was not satisfactory in terms of efficacy compared to the season (low response rate in winter) and synchrony of intervals from removal of PRID® Delta to ovulation.

Effects of a single dose HCG administration on the induction of ovulation, follicular dynamics, uterine changes and pregnancy outcomes in mares during the breeding season

Effects of a single dose HCG administration on the induction of ovulation, follicular dynamics, uterine changes and pregnancy outcomes in mares during the breeding season

Suppression of reproductive behaviour and gonadal function in female horses-An update.

The behaviour of mares is often detrimental to their performance resulting in frequent demand for methods to suppress gonadal function. In addition, prevention of unintended reproduction especially in feral horse populations may require methods for suppression of gonadal function. Surgical ovariectomy is a safe method but not an acceptable approach in feral mares and undesired in mares where future breeding is considered. There are different approaches for artificial prolongation of the luteal phase resulting in transient inhibition of oestrus and ovulation. Among those, treatment with natural or synthetic progestogens is considered the most common and successful method. Whereas application of intrauterine devices may result in prolongation of luteal function in non-pregnant mares, intrauterine insertion of glass balls is no longer recommended because of complications in individual mares. There are several safer alternatives that may be of interest, especially for population control in free-roaming horses. Treatment with long-acting deslorelin implants inhibited ovulation and oestrus behaviour in mares for limited and variable time intervals in a dose-dependent manner. The effect of GnRH vaccines varies considerably among individual mares, is age dependent, and oestrus-like behaviour may still occur. Contraception via immunization against native porcine or recombinant zona pellucida antigen is successful, but immunocontraception is as much a result of ovarian inactivity as an antibody-based block to sperm-oocyte binding. In conclusion, several treatments for suppression of gonadal function in mares are available, but there are advantages and disadvantages associated that have to be considered. The treatment of choice will thus differ with regard to the demands.

Preantral follicle population and distribution in the horse ovary.

Characterization of the ovarian preantral follicle population is a necessary step to improve understanding of folliculogenesis and ovarian physiology. Therefore, in the present study, the preantral follicle population in the equine ovary in young and old mares was investigated according to follicular morphology, follicular class, distance from the geometric center using ovarian maps, and follicular density within ovarian portions (lateral vs intermediary) and regions (dorsal vs ventral). Ovaries were collected from an abattoir and histologically processed for evaluation, and the follicle population was calculated. Overall, in the current detailed study, a higher preantral follicle population per mare ovary (mean: 82,206 ± 50,022; range: 1477 to 773,091) than originally reported was identified. Additionally, a mare age effect was observed in the follicle population (young: 152,664 vs old: 11,750) and the spatial distribution of morphologically normal and abnormal follicles and the density and population of follicular classes. These results demonstrate that, in addition to the preantral follicle population in the mare ovary being comparable to that of other species, the location and spatial distribution of these follicles is dynamic and varies depending on mare age and follicle status (i.e. morphology and developmental stage). The characterization of the distribution and population of preantral follicles in the mare ovary provided by this study can potentially aid in improving reproductive studies and assisted reproductive techniques and may expand the understanding of mechanisms involving ovarian plasticity and follicular migration.Lay summaryKnowledge of the distribution and population of immature eggs within follicles (preantral follicles) in the ovaries of mares can improve approaches to assisted reproductive techniques and fertility preservation. As the existing research on horse preantral follicle population was focused solely on large follicles, the present study provides an updated investigation of small and large preantral follicles in the mare, showing that the population is similar to those in other species. This study also shows that the way these follicles are distributed in the ovary varies depending on age and follicle characteristics. Results from this study may help to highlight which areas of the mare ovary should be looked at to find samples of good-quality follicles.