bluuebunny/crossref_metadata_2025 huggingface dataset:
11 Conception rate of high-producing dairy cows at first service using prostaglandin F2α and oestradiol benzoate
Authors: Yamaguchi M. , Takayama M. , López H. , Dochi O.
Published: December 2018

We have shown that a simplified oestrus synchronization protocol using prostaglandin F2α (PG) and oestradiol benzoate (EB) resulted in a satisfactory conception rate, similar to that obtained in high-producing dairy cows with natural oestrus (Yamaguchi et al. 2018 Reprod. Fertil. Dev. 30, 147 abstr). However, we found no significant effect of PG+EB protocols on the first-service conception rates (CR) of dairy cows. This study investigated the effect of the first-service CR by using PG+EB protocols in the high-producing dairy cows that had not exhibited oestrus expression after calving. Data concerning the first-service CR were obtained for 481 Holstein cows from 4 commercial dairy herds in Hokkaido and Ibaraki, Japan, from 2014 to 2017. The average number of parity, average interval between calving and the first service, and average milk yield (at 305 days) of the cows were 2.2±1.3, 89.7±26.1 days, and 10314±1994kg, respectively. We carried out the PG+EB protocols on cows not showing evident standing heat from Days 50 to 150 postpartum regardless of the presence of a corpus luteum in the ovary. The statuses of ovaries of cows were confirmed using ultrasonography before starting the treatment. Prostaglandin F2α (25 mg; Lutalyse, Pfizer, New York, NY, USA) was injected into cows with a >20mm cutoff for functional corpus luteum diameter at a random stage of oestrus, and EB (1 mg; Ovahormon®, Aska Animal Health Co., Tokyo, Japan) was injected 24h later, and AI was carried out 24 to 48h after EB injection. Cows with a natural oestrus service from Days 50 to 150 postpartum were used as control. All the cows were inseminated after the onset of standing heat or removal of the tail chalk. Pregnancy diagnoses were performed using ultrasonography 30 to 45 days after AI. The CR and intervals of partum to conception were analysed using a chi-square test and ANOVA. The first-service CR (i.e. the number of pregnant cows/total number of cows in the treatment group) of the natural oestrus (n=330) and PG+EB (n=151) groups were 42.7 and 41.1%, respectively, and there were no significant differences between these 2 groups (P=0.7). The average interval between calving and the first service of the PG+EB groups was significantly longer than that of the natural oestrus groups (86.4±1.5 days v. 96.9±1.9 days; P<0.01). The average days open of the natural oestrus and PG+EB groups were 88.5±3.2 days and 98.1±2.3 days, respectively (P<0.05). These results confirmed that the first-service CR in the PG+EB and natural oestrus groups of high-producing dairy cows were similar. We suggest that conducting PG+EB protocols was effective in the high-producing dairy cows that had not exhibited oestrus expression after calving. Moreover, using PG+EB protocols could prevent the further increase in the number of days open.
113 Influence of antral follicles count on conception rate in Holstein cows and antral follicles count variation on insemination day and on pregnancy Day 30 and 60
Authors: Morotti F. , Droher R. G. , Zangirolamo A. F. , Seneda M. M.
Published: December 2018

Several studies have indicated the population of antral follicles or count of antral follicles (AFC) as one of the main factors that influence the efficiency of reproductive biotechniques and its use as a marker of fertility. In this context, knowing the factors that interfere with or are influenced by this parameter is of crucial importance. Thus, the present study aimed (1) to determine the influence, in high-producing Holstein cows, of low, intermediate, and high AFC on the conception rate to AI, and (2) to evaluate if AFC shows variation at AI moment and in relation to 2 different moments of the gestation (30 and 60 days). In the first (n=95) and second (n=54) experiment, high-producing Holstein cows with a body condition score 2.5-4.5, aged 23-99 months, and maintained in a freestall system were inseminated. Using frozen semen from a single bull and performed by a single technician, the insemination was performed after detection of spontaneous estrus or induction with 25mg of dinoprost tromethamine IM. To determine AFC (follicles ≥2mm), the ovaries of each animal were examined by ultrasound at the time of AI at 30 and 60 days of pregnancy. The pregnancy diagnosis was performed by ultrasound 30 days after AI. In the second experiment, 54 females considered pregnant on the 30-day examination had the AFC reassessed 30 days later (60 days of gestation) to determine the variation in the number of follicles. In the first study, cows were divided into low (≤35 follicles, n=30), intermediate (≥40 and ≤55 follicles, n=30), and high (≥60 follicles, n=30 cows) according to the AFC quartiles (Q1, Q2, and Q3). The AFC was analysed by ANOVA followed by the Tukey's test and the conception rate by the Chi-squared test. In the second study, the AFC was compared between day of AI (considered nonpregnant), 30 and 60 days of pregnancy by repeated-measures using the generalized linear model (P ≤ 0.05). In the first study, the number of antral follicles was different (P<0.001) among groups with low (28±7 follicles), intermediate (47±5 follicles), and high AFC (72±11 follicles); however, the conception rate was similar (P=0.270) for the respective groups, at 26.7% (8/30), 36.7% (11/30), and 50% (15/30), respectively. In the second study, there was an increase (P<0.0001) in the number of antral follicles during the pregnancy, from 55.1±3.1 follicles in the AI moment to 82.6±4.2 at 30 days and reaching 115.2±5.1 follicles at 60 days of pregnancy. In conclusion, these data demonstrated that the conception rate was not influenced by the different AFC groups; however, the AFC increased as pregnancy progressed. In addition, we can suggest that the initial third of gestation (when it is still possible to manipulate the ovaries) is a strategic moment for oocyte retrieval in bovine females for the in vitro embryo production.
116 Differential expression of connexin 43 and 37 mRNA transcripts during the estrous cycle in canines
Authors: De los Reyes M. , Palomino J. , Espinoza R. , Gallego C.
Published: December 2018

Gap junctions are intercellular channels that mediate cell-to-cell communication, allowing the passage of small signalling molecules. In the ovary, connexin 43 (Cx43) and connexin 37 (Cx37) are important gap junctional proteins expressed in the granulosa and cumulus cells or oocytes of several species. Gap junctions and connexins are required for the regulation of the oocytes meiotic resumption in preovulatory follicles after the surge of LH. However, unlike other species, canine oocytes do not resume meiosis before ovulation, which could be related to expression patterns of Cx43 and Cx37 during oocyte development and ovulation. Therefore, this study aimed to address the canine Cx37 and Cx43 gene expressions throughout the oestrous cycle, including the preovulatory period. The ovaries were obtained from bitches 1-6 years old (n=72) following ovariohysterectomy. The stage of the oestrous cycle was assessed according the ovarian structures and by measurements of serum progesterone (P4) levels obtained from blood samples on the day of surgery. Anestrus was <0.1 ng/mL P4 and absence of follicles or corpus luteum in the ovarian surface; proestrus was 0.2-2 ng/mL P4 and growing small to medium follicles on the surface of the ovaries; oestrus was 2-19 mg/mL P4 and large follicles on the surface of the ovaries; and diestrus was >20 ng/mL P4 and mainly predominant corpus luteum on the ovaries. For Cx43 analysis, follicular cells (granulosa and theca) were mechanically recovered from follicles (n=620) distributed into 4 types: prenatal (1 layer of granulosa cells up to the onset of antrum formation), small antral (~0.2-0.39mm), medium antral (~0.4-5.9mm), and large antral (~6-10mm). For Cx37 study, the cumulus-oocytes complexes (COC) from the same follicles were used. Total RNA extraction was performed, and the evaluation of gene expression levels was achieved by relative quantification quantitative PCR analysis in follicular cells and COC. The data from at least 3 independent experiments for each gene were evaluated by ANOVA. The gene expression of both Connexins were observed in all stages of follicular development; however, the mRNA levels varied over the oestrous cycle. Both Cx43 and Cx37 transcripts showed the highest (P<0.05) levels at anestrus when compared to other phases. The mRNA levels of both genes remained without changes in large follicles at oestrus stage, suggesting that, in contrast to other mammals where LH down-regulates connexins expression leading to the subsequent loss of intercellular coupling, the communication between the oocyte and follicular cells was maintained in canines. In conclusion, these 2 connexin genes were differentially expressed in canine follicular cells and COC during the follicular development. The maintenance of the gene expression of these connexins at the final follicular growth may be involved in the prolonged meiotic arrest in this species.
Supported by Ga grant from FONDECYT (1171670).
117 Kit ligand enhances growth and nuclear maturation of buffalo oocytes in vitro
Authors: Islam M. N. , Alam M. H. , Khatun A. , Hashem M. A. , Moniruzzaman M.
Published: December 2018

This study aimed to investigate the effect of Kit ligand (KL), a growth factor that regulates folliculogenesis in mammalian ovaries, on growth of buffalo oocytes in early antral follicles in vitro. Cumulus-oocyte complexes were dissected from early antral follicles (1mm) of slaughtered buffaloes and cultured in Dulbecco’s minimum essential medium supplemented with fetal bovine serum, sodium pyruvate, gentamycin, hypoxanthine, dexamethasone, cysteine, polyvinylpyrolidione, l-ascorbic acid, oestradiol-17β, and androstenedione in a 96-well culture plate at 38.5°C under an atmosphere of 5% CO2 in air for 6 days. The culture medium was supplemented with 0, 50, and 100 ng/mL KL (recombinant human SCF, Cat. No. H8416, R&D Systems, Minneapolis, MN, USA). Sixty oocytes were cultured in each group with 6 replications. In vitro-grown oocytes were cultured for maturation in tissue culture medium-199 supplemented with 5% fetal bovine serum, sodium pyruvate, gentamycin, and 100 ng/mL FSH at 38.5°C for 24h under an atmosphere of 5% CO2 in air. The oocytes were then stained with aceto-orcein and examined under a differential interference contrast microscope. Data were analysed using SAS/STAT version 9.1.3 for Windows (SAS Institute Inc., Cary, NC, USA) by one-way ANOVA and means compared with Tukey’s HSD test. The mean diameter of oocytes measured at the time of seeding on the culture substrate was 100.6±0.4μm (n=180). After 6 days of culture, the diameters of oocytes increased to 110.8±0.5, 114.0±0.5, and 115.0±0.6µm in 0, 50, and 100 ng/mL KL-treated groups, respectively. The survival rates were 60.0±6, 81.2±1.2, and 92.0±4.9% in 0, 50, and 100 ng/mL KL-supplemented oocytes at Day 6. Moreover, KL pretreatment enhanced maturation of buffalo oocytes dose dependently. A small proportion of oocytes (8.4%) treated with 50 ng/mL KL reached the MII stage. This number increased to 25% when oocytes were treated with 100 ng/mL KL. These results show that KL enhances growth, viability, and meiotic progression of buffalo oocytes in vitro.
119 Combined exposure to phthalates and 4-vinylcyclohexene diepoxide accelerate the loss of ovarian follicles, leading to premature ovarian failure in rat model
Authors: Tran D. N. , Lee J.-H. , Yoo Y.-M. , Jung E.-M. , Ahn C. , Park S. Y. , Lee B. , Jeon B.-H. , Jeung E.-B.
Published: December 2018

Premature ovarian failure (POF) is observed in women under 40 years with primary or secondary amenorrhea. The causes for POF are idiopathic, genetic, iatrogenic incidence, autoimmunity, and adverse effects from toxic chemical exposures. Several classes of chemicals have been shown to alter follicle development and reduce fertility, leading to POF in mammals. We investigated the synergistic effects of 4-vinylcyclohexene diepoxide (VCD) and phthalates, including di(2-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP), on POF. Both VCD and these phthalates have been reported to disrupt the folliculogenesis and steroidogenesis in many species. In the vehicle group, female Sprague-Dawley rats (8 weeks of age) received an IP injection of corn oil daily for 2 weeks. The 7 other groups received VCD+corn oil, DEHP, DEHP+VCD, BBP, BBP+VCD, DBP, or DBP+VCD. After receiving the VCD or vehicle injection IP for 2 weeks, corn oil or a phthalate were orally administered once a day for the entire 6-week study period. The mRNA expressions of Amh and Sohlh2 were significantly decreased in the combination groups compared to the control and individual groups. Serum Amh levels were significantly lower in the combination groups. Additionally, serum levels of FSH were markedly higher in combination groups. Our findings suggest that the combination of VCD and phthalates can accelerate the POF by disturbance in folliculogenesis and hormone regulation.
121 Ovulatory response to gonadotropin-releasing hormone relative to day and diameter of dominant follicle during the first follicular wave in Beetal goats
Authors: Ahmad W. , Khan M. I.-R. , Murtaza A. , Mohsin I. , Riaz A. , Javed K.
Published: December 2018

In Beetal goats under subtropical conditions, ovulatory response of first-wave dominant follicle based on diameter and day of oestrous cycle is unknown to date. The objective of this study was to evaluate the ovulatory response to gonadotropin-releasing hormone (GnRH) relative to day and diameter of dominant follicle during the first follicular wave in Beetal goats. Fifty goats were synchronized using a single dose of prostaglandin F2α (75 µg; D+ cloprostenol, Fatro, Italy), and scanned every 6 hours for ovulation using a 7.5-MHz trans-rectal transducer (Honda HS: 1500, Japan). Following ovulation (Day 0), goats (n=40; 10/day) were given GnRH (25µg lecirelin, Fatro, Italy) on Days 2, 4, 6, and 8 of oestrous cycle. Follicular dynamics was monitored daily from D 0 until GnRH, and every 4 hours after GnRH until ovulation. Ovulation frequency relative to day and dominant follicular diameter was analysed by binary logistic regression, whereas diameter of dominant follicle at time of GnRH, ovulation time, time of wave emergence and growth rate of dominant follicle following GnRH were analysed by one-way ANOVA and independent sample t-test (SPSS 20, IBM Corp., Armonk, NY, USA). Eighty percent of the goats ovulated within 74.7±1.3h after prostaglandin F2α. Overall, 37% (14/38) of the goats ovulated following GnRH within 12.0±1.5h. Ovulations (%) following GnRH were higher (P=0.03) on Day 4 than 2 (60 v. 10%; i.e. 14 times higher than Day 2) and decreased gradually from Day 4 to 8 (P>0.05). Similarly, ovulations following GnRH were higher (P=0.00) in first-wave dominant follicles having diameter 6.1-7.0mm than those having less than 6.1mm (88v. 23%; i.e. 24 times higher than <6.1mm). Ovulations (%) decreased as the diameter of dominant follicle increased from 6.1-7.0mm (P>0.05). Diameters of ovulatory follicles at time of GnRH between Day 2, 4, 6, and 8 were not different (P>0.05). Regardless of day of GnRH treatment, diameter of ovulatory v. anovulatory follicles differed (6.44±0.03v. 5.1±0.4 mm; P=0.00). Overall, growth rate of dominant follicles following GnRH was 0.2±0.11mm and did not differ between ovulatory and anovulatory goats (0.29±0.13v. 0.13±0.15 mm; P>0.05). Interval to the emergence of a new wave following GnRH was prolonged for Day 4 compared to Day 2, 6, and 8 (i.e. 22v. 8, 11, and 12 h; respectively, P<0.05). In conclusion, GnRH treatment at Day 4 resulted in maximum ovulations as the first-wave dominant follicle reached a size of 6.1 to 7.0 mm.
122 Effect of gonadotropin-releasing hormone plus 7-day progesterone (CIDR) with or without eCG on follicular dynamics, estrus response, and pregnancy rate in anestrous Beetal goats during nonbreeding season under subtropical conditions
Authors: Khan M. I.-R. , Hameed N. , Ahmad W. , Abbas M. , Murtaza A. , Ahmad N. , Javed K.
Published: December 2018

Oestrus induction is a challenging task during the nonbreeding season in goats. Therefore, our objective was to evaluate the efficacy of gonadotropin-releasing hormone (GnRH) plus a 7-day CIDR-based protocol with or without eCG for the induction of oestrus and fertility in acyclic Beetal goats during the nonbreeding season under subtropical conditions. Based on the absence of corpus luteum via ultrasonography, goats (n=56; mean weight=32.8±0.5 kg; body condition scre=2.5±0.1) were inserted with a CIDR (300mg progesterone) along with GnRH (Day 0), and randomly treated either with eCG (400 IU; n=35) or without eCG (control; n=21) on Day 7. The CIDR was removed on Day 7 and all does were administered single dose of prostaglandin F2α (75µg cloprostenol; Fatro, Italy). Following CIDR insertion, ovarian follicular dynamics was monitored daily through a 7.5-MHz transrectal transducer (Honda HS 1500, Japan) until ovulation in a subset of eCG (n=11) and control goats (n=10). Oestrus was detected at every 12h after CIDR removal with aproned bucks and does were bred naturally. Pregnancy was detected 25 days after breeding by transrectal ultrasound. Data were analysed by t-test, ANOVA, and Chi-squared test using SPSS (version 20.0, SPSS Inc., Chicago, IL, USA). Oestrus occurred more often following CIDR removal in eCG v. control goats (94v. 57%; P<0.05). Similarly, onset of oestrus was earlier (36.0±2.1v. 54.2±4.7 h; P<0.05) and the duration of oestrus was longer in eCG v. control goats (i.e. 30.2±1.3v. 22.1±2.3 h; P<0.05). However, diameter of preovulatory follicles and interval to ovulation after CIDR removal were similar between eCG and control goats (P>0.05). In contrast, growth rate of preovulatory follicle after CIDR removal until ovulation in eCG goats was greater, and ovulation rate was higher than control goats (0.7±0.1v. 0.3±0.1 mm/day; 2.2±0.2v. 1.4±0.2, respectively; P<0.05). In eCG goats, the dominant follicle grew rapidly between Days 8 to 9 (P<0.05), whereas within control goats the dominant follicle grew at consistent rate (P>0.05). After CIDR removal, percentage of ovulating goats in eCG was higher as compared to the control group (100v. 44%; P<0.05). Within control goats, size of the dominant follicle in ovulatory goats was greater at the time of CIDR removal than anovulatory goats (5.7±0.3v. 5.0±0.15 mm; P<0.05), whereas wave emergence tended to be earlier after CIDR insertion in anovulatory goats (4.2±1v. 2.6±0.4 days; P=0.09). Pregnancy rate 25 days postbreeding did not differ between eCG and control goats [i.e. 49% (16/33) v. 25% (3/12); odds ratio 2.9/1; P>0.05]. In conclusion, the eCG protocol resulted in higher oestrus response, ovulation rate, and 3 times higher pregnancy rate in anestrous goats during the nonbreeding season.
123 Effect of heat stress on oocyte developmental competence and global gene expression dynamics in Bos taurus crossbred beef cows
Authors: Jiang Z. , Diaz F. A. , Gutierrez E. J. , Foster B. A. , Hardin P. T. , Bondioli K. R.
Published: December 2018

It is known that animals under the effect of heat stress present reduced fertility. We aimed to investigate the effect of heat stress on the developmental competence and global gene expression profile of oocytes through the transition from spring to summer under Louisiana conditions. Oocytes were collected from 6 crossbred, non-lactating cows once a month from May to July. Temperature and humidity indexes for May and July were 72.48 and 78.06, respectively. An index above 75 indicates that cows are under heat stress. All cows underwent dominant follicle removal, and then 7 days later, ovum pickup was performed to aspirate germinal vesicle (GV) oocytes. Half of the oocytes were processed for RNA-seq as GV, and half were matured in vitro to metaphase II (MII). Smart-seq protocol was followed to prepare RNA sequencing libraries from a pool of 4 oocytes (GV n=6; MII n=6). Sequencing reads were pre-filtered and aligned to the bovine genome, and gene expression values were calculated as transcripts per million. Genes were deemed differentially expressed between different conditions if they showed a false discovery rate P-value<0.05 using DESEqn 2 package. DAVID (https://david.ncifcrf.gov) and IPA (ingenuity pathway analysis) were used to reveal the gene ontology and pathways, respectively. The RNA sequencing showed that a total of 212 genes were differentially expressed as a result of heat stress at the GV stage, with 94 and 118 genes up- and down-regulated, respectively. Gene ontology analysis indicated significant over-representation of elements involved in steroid biosynthetic process, oxidation reduction, and mitophagy in response to mitochondrial depolarization. Several pathways were influenced by heat stress, including glucocorticoid biosynthesis, apoptosis signalling, and HIPPO signalling. At the MII oocyte stage, only 93 genes (19 up-regulated and 74 down-regulated) were significantly differentially expressed in oocytes between July and May groups. Oocytes retrieved on different collection days, from the same cows under the same treatments, showed no difference on maturation rates, suggesting that the in vitro maturation process equalizes the expression of several genes. The primary biological processes significantly affected in MII oocytes were regulation of MAPK cascade, melanosome organisation, and negative regulation of transcription. In addition, we found that UBE2I, a gene involved in phosphorylation-dependent sumoylation of heat shock factor 1 (HSF1), was significantly up-regulated in July compared with May in MII oocytes. Interestingly, only 5 common genes were significantly affected by heat in both GV and MII oocytes: E2F8, GATAD2B, BHLHE41, FBXO44, and RAB39B. Our findings provide new insight into the molecular mechanisms of detrimental conditions (heat stress) on bovine oocytes, which may help to reveal master regulators controlling oocyte competence.
124 Functional analysis of porcine OCT4 transcriptional regulatory region-based reporter system
Authors: Kim S.-H. , Choi K.-H. , Lee D.-K. , Lee M. , Cho M.-H. , Oh J.-N. , Lee C.-K.
Published: December 2018

Gene OCT4 plays pivotal roles in maintaining pluripotency of early mammalian embryonic development and embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region for the study of pluripotency. However, there is still a lack of sufficient information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, first, we conducted an investigation to find conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. A similarity of nucleotide sequences of the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and 4 regulatory regions including distal and proximal enhancer elements have a high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay indicated that the porcine OCT4 distal enhancer and proximal enhancer are highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively (n=3). Comparison analysis of naïve (Tbx3, Nr0b1, Rex1, Esrrb, Nanog, Klf2) or primed (Gata6, Mixl1, Fgf5, Otx2) state marker gene expression in a dual-reporter assay using pOCT4-DE-eGFP and pOCT4-PE-DsRed2 showed that expression of naïve and primed markers were up-regulated in cells with high green fluorescent protein and red fluorescent protein expression, respectively (n=3). Porcine OCT4-upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work could provide basic information for the porcine OCT4 upstream region and the various porcine OCT4-fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and for the establishment of embryonic stem cells in pigs.
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Development of High Value-Added Food Technology Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA, 118042-03-1-HD020).
129 Knockdown of connexin 43 effects on developmental competence in porcine parthenotes
Authors: Shin K.-T. , Niu Y.-J. , Nie Z.-W. , Zhou W. , Kim Y.-H. , Cui X.-S.
Published: December 2018

Connexin 43 (Cx43) is one of the gap junction proteins that are compounds of transmembrane proteins and transports the small-molecular-weight chemicals up to 2 kDa. Lacking of Cx43 influences the junctional protein and induces autophagy and apoptosis in somatic cells. However, the function of Cx43 in porcine early embryos is still unknown. With the aim to find out the molecular mechanism of Cx43 on the developmental competence of early porcine embryos, dsRNA technology was carried out and inhibitor was used as the positive control. Additionally, it is difficult to obtain pig embryos of homogeneous quality due to the relatively high incidence of polyspermy during IVF. Therefore, diploid parthenotes have been frequently used to study early development in the pig. Connexin 43 dsRNA (1μg μL−1) was microinjected into the parthenogenetically activated porcine zygotes. Blastocyst rate [5 repeats; treatment, 8.8±1.6% (n=1356) v. control, 38.6±4.3% (n=1082)] and total cell numbers in the blastocyst [4 repeats; treatment, 20.7±3.5 (n=85) v. control, 39.8±4.1(n=97)] were significantly reduced following Cx43 knocking down. Results from fluorescein isothiocyanate-dextran and Western blot assay show that knockdown (KD) of Cx43 significantly increased membrane permeability through down-regulation of genes that are components of both adherence and tight junction in the porcine blastocyst. Reactive oxygen species were significantly increased in the Cx43 KD group compared with the control. In addition, KD of Cx43 activated Caspase 3 and significantly increased ATG8 expression and induced autophagy and apoptosis. Results suggest that KD of Cx43 influences pre-implantation porcine embryo development via increasing membrane permeability and reactive oxygen species generation and by inducing autophagy and apoptosis.
13 Evaluation of PG-3-G presynchronization in Ovsynch-P programs in lactating dairy cows: Preliminary results
Authors: Velho G. S. , Oliveira C. R. , Berlitz C. G. B. , Luiz D. S. V. , Chaiben M. F. C. , Dalto A. G. C. , Borges J. B. S.
Published: December 2018

Presynchronization treatments associated with the Ovsynch protocol and its modifications have been used to improve reproductive performance in lactating dairy cows. Use of a PG-3-G protocol is indicated to increase the number of cows in the most ideal stage of the oestrous cycle (Days 5-9) at first gonadotropin-releasing hormone (GnRH) injection of the Ovsynch protocol. The objective of this study was to compare fertility of lactating dairy cows submitted to Ovsynch-P, preceded by PG-3-G protocol or not. A total of 86 Holstein cows [body condition score of 2.6±0.2 (1-to-5 scale), 3 and 10 years old, mean milk yield of 28.1±7.9kg per day, and 118±56 days postpartum period] maintained in a semi-intensive system were blocked by parity and date of calving and randomly assigned to groups: control (n=44) and PG-3-G (n=42). On Day -10 cows in the PG-3-G group received an injection of 500µg of sodium cloprostenol IM (Estron, Agener União, Brazil), and on Day -7 they received 0.05mg of Lecirelin IM (GnRH, Gestran, Agener União). On Day 0, all cows had the first dose of Ovsynch GnRH injection and a 1-g progesterone intravaginal implant (P4, Primer, Agener União). On Day 7, a dose of sodium cloprostenol was administered to induce corpus luteum (CL) luteolysis, and the P4 implant was removed. Forty-eight hours later, cows were injected with the second dose of GnRH and inseminated 12 to 18h later. The presence of CL and ovulatory follicle (OF) diameter were determined by transrectal ultrasonography (Aquila, Pie Medical, Maastricht, the Netherlands) on Days 0, 7, and 9, and gestation diagnosis was done on Day 30 after AI. Statistical analysis was performed using ANOVA, chi-square, Mann-Whitney, and Pearson correlation tests in SPSS 18 (Armonk, NY, USA), considering P=0.05 significant and a tendency when P>0.05 but P=0.10. Incidence of CL on Day 0 tended to be more frequent (P=0.1) in the PG-3-G group (78.6%) compared with the control (59%), but CL presence on Day 7 did not differ (P=0.98) between the groups (35 and 34%, respectively). Based on the odds ratio, PG-3-G cows were 2.16 times more likely to have a CL on Day 0. The mean diameter of OF on Day 9 was smaller (P<0.05) in PG-3-G cows (12.6mm) than in control cows (15.3mm), and a negative correlation was detected (r=−0.29) between the presence of CL on Day 0 and the diameter of OF on Day 9 (P=0.05). Pregnancies per AI tended (P=0.08) to be higher in PG-3-G cows (52%) than in control cows (34%). In summary, cows submitted to presynchronization with the PG-3-G protocol presented higher frequency of CL on Day 0 and tended to have a higher pregnancies per AI compared with control cows.
134 The effect of follicle-stimulating hormone and equine chorionic gonadotropin injection protocols on laparoscopic ovum pickup in prepubertal Kiko goats
Authors: McElyea D. , Carwell B. , Carwell D. , Kennedy D. , Looney C. , Steinhauser C.
Published: December 2018

In vitro fertilization has served as a great tool in the human and bovine field in its ability to treat infertility in females. The purpose of this study was to compare hormone injection treatments in goats using the laparoscopic ovum pickup technique. A crossover trial was conducted to evaluate 2 superovulation injection protocols [a multiple-injection FSH (F) and a one-injection FSH-eCG (F+E)] for their ability to increase recovery of oocytes using laparoscopic ovum pickup (LOPU) technique in 10 prepubertal Kiko does. Does were randomly assigned to 2 sequence groups (n=5). Period 1 initiated on 28 July 2017 and Period 2 on 12 August 2017. A CIDR device was inserted on Day 0 and removed after LOPU. On Day 6 of each period, 3mL of prostaglandin F2α was given IM to each doe. F-treated does received (IM) twice daily injections of 15, 12.5, 10, and 7.5mg of FSH, whereas F+E-treated does received (IM) 80mg of FSH and 300 international units (IU) of eCG. Thirty-six hours after injection, follicles were aspirated using LOPU. Treatment response measurements included total follicles observed, oocyte recovery rate, and blastocyst rate. Total follicles observed were higher (P=0.004) in F+E-treated does (305 follicles) than F-treated does (236 follicles). Neither oocyte recovery rates (F+E=41.5% and F=38.1%) nor blastocysts rates (F+E=18% and F=32%) were affected (P>0.05) by injection protocol. In conclusion, we summarise that multiple injections of FSH can results in a higher number of follicles produced; however, this does not have a subsequent effect on IVF and development rates.
135 Use and dose of porcine follicle-stimulating hormone for ovarian superstimulation prior to ovum pickup and in vitro embryo production in pregnant Holstein heifers
Authors: Sala R. V. , Carrenho-Sala L. C. , Fosado M. , Peralta E. , Pereira D. C. , Moreno D. , Moreno J. F. , Garcia-Guerra A.
Published: December 2018

The benefit of superstimulation with exogenous FSH before ovum pickup for in vitro embryo production has been the subject of significant controversy. In addition, there is limited information on different dose regimens. Thus, the objective of the present study was to evaluate the effect of dose of porcine (p)-FSH during superstimulation before ovum pickup (OPU) on in vitro embryo production in pregnant heifers. Pregnant Holstein heifers (n=36) were assigned to a complete 3×3 crossover design. Three treatment groups were evaluated as follows: p-FSH 0mg (FSH0), p-FSH 160mg (FSH160) and p-FSH 300mg (FSH300). Three sessions of OPU were performed on each animal at 48, 62 and 76 days of gestation, with a washout interval between sessions of 14 days. Follicular wave emergence was synchronized by dominant follicle removal. Heifers in the FSH0 group received no further treatment, whereas the remaining groups received a total of 4 injections 12h apart as follows: FSH160 (48.0, 42.7, 37.3 and 32.0mg) or FSH300 (90.0, 80.0, 70.0 and 60.0mg), beginning 36h after dominant follicle removal. Ovum pickup was performed in all heifers 40h after the last p-FSH injection. Heifers were subjected to OPU for oocyte recovery, and number of follicles was determined. Recovered oocytes were processed and in vitro embryo production performed. Differences between treatment groups were evaluated by generalized linear mixed models. Data are presented (Table 1) as mean±standard error of the mean. There was no effect of days in gestation for any of the outcomes evaluated (P>0.05). Follicle numbers at the time of oocyte recovery were different (P<0.01) between groups. Heifers in the FSH300 group had a greater (P<0.05) number of medium, large and total follicles than heifers in the FSH0 group, whereas heifers in the FSH160 were intermediate. Total number of recovered, viable and cleaved oocytes were greater (P<0.01) in FSH300- than in FSH160- and FSH0-treated heifers. Cleavage rate and blastocyst development rate were not different (P>0.10) between groups. The number of grade 1 and 2 blastocysts was greater in FSH300- than in FSH160- and FSH0-treated heifers (P<0.03). In summary, the use of 300mg of p-FSH before OPU in pregnant heifers increases the number of follicles, oocytes and blastocysts produced per heifer with no detrimental effect on oocyte competence.

Table 1.Ovum pickup and in vitro embryo production in pregnant heifers treated with different doses of porcine FSH
136 Efficient in vitro embryo production system using in vivo-matured oocytes from superstimulated Japanese black cows
Authors: Egashira J. , Tatemoto H. , Wada Y. , Yamanaka K.
Published: December 2018

In this study, we examined whether in vivo matured oocytes collected by ovum pickup (OPU) from superstimulated Japanese black cows can improve the productivity and quality of in vitro-produced embryos. Cows in the stimulated group received an intravaginal progesterone-releasing device (Day 0), administration of 100μg of GnRH on Day 5, a single administration of 30 Armour units of FSH on the evening of Day 8 and prostaglandin F2α on the evening of Day 10. The progesterone device was removed on the morning of Day11, and then 100μg of GnRH was administered on the morning of Day 12 (0 h). The OPU and IVF were conducted at 25~26 and 30h, respectively. Cows in the control group received no treatment before OPU, and collected oocytes were subjected to in vitro maturation followed by IVF. The cortical granules distribution of oocytes at metaphase II stage, the cleavage pattern of embryos at the first cell cycle, the developmental rate, and the quality of blastocysts were compared between the stimulated and control groups. Oocytes with cortical granules distributing cortical cytoplasm were classified as normal distribution. The cleavage pattern was evaluated at 28h after IVF as follows: embryos with blastomeres of the same size without fragmentation were classified as normal cleavage; embryos with 2 blastomeres and several small fragments, direct cleavage from the one-cell stage to 3 or 4 blastomeres, or 2 blastomeres of different size were classified as abnormal cleavage. The developmental rate to blastocyst stage was measured on Day 9 of culture. The morphological quality of blastocysts was evaluated based on the IETS manual. All data were obtained from more than 3 replicates. In vitro development and cortical granules distribution data were analysed using chi-squared test. Other data were analysed using Student’s t-test. Normal cortical granules distribution rate in the stimulated group was higher than that in the control group (90.3v. 23.1%; P<0.01). Although no differences in the developmental rate to blastocyst stage (51.5v. 58.6%) was observed, the normal cleavage rate (73.4v. 51.2%) and the transferable embryo rate (98.3v. 88.0%) in the stimulated group were significantly higher (P<0.01) than those in the control group. The ratio of embryos from normal cleavage among the transferable embryos in the stimulated group was also significantly higher than in the control group (82.2v. 57.2%; P<0.01). In addition, the freezable embryo ratio (71.7v. 58.1%; P<0.072) and the total production number of embryos per head (28.0v. 15.5; P<0.106) showed a tendency to be higher in the stimulated group than in the control group. These results suggest that high quality embryos can be efficiently produced by the use of in vivo matured oocytes collected by OPU from superstimulated Japanese black cows.
138 Effects of different treatments of donors on the efficiency of embryo production and conception in bovine ovum pickup-in vitro production
Authors: Katae A. , Kaneda Y. , Sugawara M. , Nishisouzu T. , Dochi O. , Imai K.
Published: December 2018

An in vitro-produced bovine embryo has a low conception rate compared with that of an in vivo embryo. The present study was conducted to examine the effects of different treatments delivered to donors before ovum pickup (OPU) sessions to improve the conception rate of in vitro-produced bovine embryos. In total, 351 OPU sessions were performed on 138 Holstein and 213 Japanese Black cows from January to December 2017. Donors were divided into 4 groups based on their pretreatment before OPU: (1) single injection of 2.5 AU of FSH 40h before OPU; (2) CIDR insertion on Day 0, injection of 2mg of oestradiol benzoate on Day 1, 4 injections of FSH (each 2.5 AU) every 12h beginning from Day 5 to 7, followed by removal of CIDR and OPU on Day 9; (3) injection of 50μg of gonadotropin-releasing hormone 72h before OPU; or (4) no pretreatment. The collected cumulus-oocyte complexes were matured for 22h in 25mM of HEPES buffered TCM-199 supplemented with 5% newborn calf serum and 0.02 AU mL−1 FSH. After 6h of gamete co-culture (5.0×106 sperm mL−1), the presumptive zygotes were washed and the remaining cumulus cells were denuded by pipetting. The presumptive zygotes were then cultured in KSOMaa supplemented with 5% newborn calf serum for 9 days in a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). Blastocyst formation rates were analysed 9 days after insemination, and the formed blastocysts were transferred to oestrous synchronized recipients on the seventh or eighth day after oestrus. The data were analysed by Chi-squared test with Yates correction. The average numbers of collected oocytes were 57.7±17.4 (n=136), 25.3±12.8 (n=20), 28.8±12.5 (n=18) and 24.3±12.9 (n=177) in groups 1 to 4, respectively. Groups 1 and 2 showed significantly (P<0.01) high percentages of Grade-1 oocytes (52.1 and 49.6%, respectively) compared with groups 3 and 4 (37.3 and 39.9%, respectively). The proportion of blastocysts in groups 1 (38.6%) was significantly different compared with that in groups 2 (32.1%) and 4 (35.3%), but the difference was insignificant in the case of group 3 (36.4%). The conception rates in groups 1 (43.5%, n=868) and 2 (59.1%, n=44) were significantly (P<0.05) higher than those in groups 3 (35.1%, n=57) and 4 (34.9%, n=768). These results suggest that although the efficiency of embryo production did not differ largely between donors pretreated with 4 FSH injections and those without any pretreatment, the conception rate in donors pretreated with 4 FSH injections was significantly higher than that in donors without pretreatment. Moreover, donors pretreated with a single injection of FSH showed significantly high efficiency of embryo production and conception rate than donors without pretreatment.
139 Effect of protein and calcium ionophore A23187 concentration on hyperactivated motility and acrosome status of stallion sperm
Authors: Ortiz I. , Resende H. , Felix M. , Love C. , Hinrichs K.
Published: December 2018

In vitro fertilization does not occur readily in the horse. Fertilization can be achieved using sperm treated with the calcium ionophore A23187 (CaI), but rates are low and variable. In order to fertilize, it is thought that the sperm must show hyperactivated motility and undergo the acrosome reaction. The presence of protein in the media is thought to suppress the effect of CaI, but protein is needed for maintenance of sperm motility. Therefore, the objective of this study was to assess the effect of CaI in the presence or absence of protein on the acrosome and on hyperactivated motility of equine sperm. For this purpose, sperm from 4 stallions were exposed for 10min at 37°C to vehicle or to 1 (C1), 5 (C5) or 10 (C10) μM CaI, with (BSA) or without (N) 7mg mL−1 BSA. The sperm were then washed and incubated at 37°C for 2h. Total and hyperactivated motility were measured by computer-assisted semen analysis. Sperm were considered hyperactivated if curvilinear velocity was >180μm s−1, amplitude of lateral head displacement was >12μm, linearity was <30% and fractal dimension value was >1.3. The percentage of live acrosome-reacted sperm was measured by flow cytometry after staining with propidium iodide and Pisum sativum agglutinin. Data were analysed by repeated-measures 2-way ANOVA. Results were expressed as mean±standard error. Total motility in C5 and C10 treatments was significantly decreased in relation to control (BSA-vehicle) starting at 30min of incubation (35.42±13.57 to 28.20±13.10% v. 71.72±9.21%, respectively; P<0.05). Hyperactivated motility was significantly lower in C10, C5 and N-C1 than in control after 2h of incubation (1.46±0.64v. 3.10±0.58%, respectively). Live acrosome-reacted sperm were significantly higher (P<0.05) for BSA-C5 (14.04±1.99%) and BSA-C10 (14.85±2.52%) than for control (7.50±1.62%) after 2h of incubation. The exposure to sperm of concentrations ≥5μM CaI was associated with loss of motility from 30min of incubation on. However, 2h of incubation after ≥5 μM CaI in the presence of BSA were needed to increase the percentage of live acrosome-reacted sperm. This mismatch between motility and acrosome response helps to clarify the reasons for the variable effect of sperm CaI treatment on equine IVF. Further studies measuring calcium influx and assessing the effect of sperm pre-incubation on CaI response are needed to explore mechanisms for equine in vitro sperm capacitation.
140 A comparison of in vitro embryo production between heifers and lactating Holstein donors without superstimulation
Authors: Silva E. P. , Sermersheim M. K. , Marchioretto P. V. , Della Mea R. , Naves L. M. , Rivelli I. , Rochelle F. , Nasser L. F. T. , Rubessa M. , Wheeler M. B.
Published: December 2018

Bovine oocyte donor age seems to have an important role on in vitro embryo production. Therefore, the aim of this study was to compare cumulus-oocyte complex (COC) recovery, cleavage, and blastocyst rates between heifers and lactating Holstein donors. A total of 89 animals (heifers: n=60, 11 to 17 months of age; lactating cows: n=29, 60 to 180 days in milk) were used in this experiment. Ovum pickup (Day −1) was performed on a random day of the oestrous cycle without superstimulation by a single technician. Conventional or sexed semen were used for IVF (Day 0). Cleavage and blastocyst rates were evaluated on Days 3 and 7, respectively. Cleavage was determined when structures had 4 or more cells and blastocyst rate included grades I and II blastocysts. Continuous data were analysed by t-test, and binomial data were analysed by Fischer’s exact test. A total of 1,289 COC were recovered from lactating cows (n=509) and heifers (n=777). Total and viable COC per donor were higher (P<0.05) in lactating cows (total=17.55±13.08; viable=11.24±8.9) than heifers (total=12.95±5.39; viable=7.88±4.08). The percentage of viable COC was similar (P>0.05) in lactating cows (64.04%; 326/509) and heifers (60.87%; 473/777). Cleavage rate was higher (P<0.0001) in lactating cows (58.74%; 299/509) than in heifers (45.05%; 350/427). Cleavage rate was similar (P>0.05) with conventional and sexed semen in heifers (conventional=37.68%; 52/138; sexed=46.63%; 298/639) and cows (conventional=55.05%; 49/89; sexed=59.52%; 250/470). Blastocyst rate was higher (P<0.0001) in lactating cows (19.84%; 101/509) than in heifers (8.49%; 66/777). Conventional semen had a higher (P<0.0001) blastocyst rate (18.11%; 24/138) than sexed semen (6.57%; 42/639) in heifers. However, there was no difference (P>0.05) in blastocyst rate between conventional (24.71%; 22/89) and sexed semen (18.8%; 79/420) in lactating cows. Blastocyst/cleaved structures ratio was higher (P<0.05) in heifers using conventional (46.15%; 24/52) compared with sexed (14.06%; 42/298) semen. However, in lactating cows there was a tendency (P<0.1) for higher blastocyst/cleaved structures ratio using conventional (44.89%; 22/49) compared with sexed semen (31.6%; 79/250). In conclusion, a higher number of total and viable COC were aspirated from Holstein lactating cows than heifers. Furthermore, cleavage and blastocyst rates were higher in lactating cows than in heifers. Conventional semen gave more embryos than did sexed semen in heifers but not in cows. Overall, oocytes from Holstein lactating donors are more suitable for in vitro embryo production than are those from heifer donors.
142 Effect of follicular size on oocyte recovery rate, quality, and in vitro developmental competency following maturation, fertilization, and culturing in Bos indicus cows
Authors: Sarwar Z. , Saad M. , Saleem M. , Riaz A. , Ahmad N.
Published: December 2018

Applied reproductive technologies include AI, multiple ovulation and embryo transfer, IVF, cloning, ovum pickup, and zygote intra-fallopian tube transfer in farm animals. Modern commercialized dairy farming is ever more relying on in vitro embryo production for maximal utilisation of superior genetics of female animals. Follicular size influenced the quality of the oocytes and their in vitro developmental competence in Bos taurus cows. However, this information is lacking in Bos indicus cows. Therefore, the objectives of the present study were to determine the effect of follicular size on recovery rate, quality, and in vitro developmental competence of the oocytes in Bos indicus cows. The ovaries (n=508) of 5- to 8-years-old Bos indicus cows, body condition score 2.75±0.25, having mixed parity and clinically normal reproductive tracts, were collected. These follicles on the ovaries were divided into 2 groups based on their size: (1) <6mm diameter and (2) ≥6mm diameter. The data on recovery; quality; maturation; cleavage rates; 4-cell, 8-cell, and 16-cell stages; and morula rate were analysed by Chi-squared test using SPSS version 20 software (IBM Corp., Armonk, NY, USA, 2016) system for windows. Results revealed that oocytes with grade I+II quality (68.4v. 42.1%; P<0.05) were higher, whereas grade III+IV quality (31.6v. 57.5%; P<0.05) were lower, in ≥6mm as compared with <6mm groups, respectively. Moreover, the 4-cell (34.5v. 18.7%), 8-cell (15.4v. 7.7%), 16-cell (8.7v. 2.2%) stages and morula rate (4.5v. 0.3%) were higher (P<0.05) in ≥6mm as compared with <6mm groups, respectively. However, the maturation and cleavage rates did not differ (P>0.05) between both groups. It is concluded that oocytes having follicle size ≥6mm have better quality in terms of morphological grades and in vitro developmental competence in Bos indicus cows. It is implied that for maximum production of embryos through ovum pickup, the most appropriate follicular size for aspiration is ≥6 mm.