231 EFFECTS OF DIETARY PROPYLENE GLYCOL ON FOLLICULAR FLUID AND OVUM PICK UP IN VITRO EMBRYO PRODUCTION IN GROWTH-RESTRICTED HEIFERS WITH TWO PROFILES OF ANTI-MÜLLERIAN HORMONE

Fertility and embryo quality can be improved in cattle by using diets that induce a programmed modulation of circulating insulin concentrations. The aim of this study was to test whether the daily oral administration of propylene glycol (PG) could modify metabolite and hormone plasma and follicular fluid concentrations and improve in vitro embryo production in superovulated growth-restricted heifers (600 g day–1). Sixteen Holstein heifers were grouped according to their pre-experimental anti-Müllerian hormone (AMH) plasma concentrations: low (L = 1–80 pg mL–1; n = 7) or high (H: >150 pg mL–1; n = 9). Heifers received a single daily drench from Day 1 to Day 9 of an oestrous cycle [first cycle, 400 mL of water (control) and second cycle, 400 mL of PG]. Serial jugular blood samples were collected on Day 7 of each cycle to monitor plasma insulin, glucose, and β-hydroxybutyrate (BHB) concentrations in relation to the drench. Blood samples were also collected to measure insulin-like growth factor-1 (IGF1) and progesterone (P4) concentrations on Days 0, 2, 5, 7, and 9 of the oestrous cycle. Follicular fluid was collected on Day 9 to measure insulin and IGF1 concentrations. Ovarian ultrasonography was performed on Days 2 and 5 to count follicles between 2 and 8 mm in diameter and estimate their size. After ovum pickup (OPU) performed following superovulation on Day 5 of the oestrous cycle, oocytes were matured and fertilized in vitro, then embryos were cultured for 7 days. Propylene glycol increased plasma concentrations of insulin and glucose and reduced BHB in both groups of heifers compared with control. It also increased IGF1 concentrations on Days 5 and 7 in AMH L heifers and on Days 2, 5, and 7 in AMH H heifers, and reduced P4 concentrations on Days 5 and 9 of the oestrous cycle in all heifers. In follicular fluid, there was no difference in insulin concentrations between groups, but PG increased IGF1 concentrations in all heifers. In ovaries, PG increased the number of small follicles (2–3 mm) and total follicles on Day 2 of the cycle in all heifers, and medium follicles (4–8 mm) and total follicles on Day 5 in AMH H heifers. Propylene glycol improved the in vitro embryo development rate (total number of embryos/number of fertilized oocytes) in all heifers (AMH L: control, 37.9% v. PG, 50.0%; P < 0.05; AMH H: control, 36.4% v. PG, 48.3%; P < 0.05). In AMH H, the number of grade 1 blastocysts was increased by PG (control, 5.2 ± 1.0 v. PG, 8.9 ± 1.0; P < 0.01), whereas there was no difference between treatments in AMH L heifers (control, 1.9 ± 1.1 v. PG, 3.2 ± 1.1; P > 0.05). These results indicate that short-term oral PG supplementation affects the concentrations of metabolites and metabolic hormones in blood and IGF1 concentrations in follicular fluid. PG administration is effective in improving in vitro embryo production more markedly in heifers with high AMH compared with low AMH endocrine levels.

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Effects of Various Levels of Luteinizing Hormone and Caprine Follicular Fluid on In Vitro Embryo Production of Shami Goat

ISPEC Journal of Agricultural Sciences ◽

10.46291/ispecjasvol5iss3pp575-584 ◽

2021 ◽

Vol 5 (3) ◽

pp. 575-584

Author(s):

Omar Mardenli ◽

Hadi Awad Hassooni ◽

Mahdi Saleh Mohammad Al-Kerwi

Keyword(s):

Luteinizing Hormone ◽

Follicular Fluid ◽

Culture Medium ◽

Goat Breed ◽

Embryo Production ◽

Follicle Size ◽

In Vitro Embryo Production ◽

Type 3

In the current study, the hypothesis of the effects of luteinizing hormone (LH) and follicular fluid (FF) derived from follicles of varying size on in vitro embryo production of the Shami goat breed were tested. The caprine follicular fluid (cFF) was obtained from healthy female’s ovaries by aspiration method and classified into two main classes (follicles with a diameter of ≤ 2mm and ≥3mm). The resulting cFF was added to the culture medium TCM-199 through six Treatments (A, B and C with a source of follicle size of ≤ 2mm; D, E and F with a source of size of ≥3mm). LH was added only to four of the previous Treatments with the levels of 50 µg ml-1 (B and E) and100 µg ml-1 (C and F). Results of the study showed that the oocytes incubated in Treatment F achieved a clear superiority (p=0.001) in the rates of maturation (87.0%), fertilization (80.0%) and cleavage (82.3%). The oocytes incubated in the same Treatment (F) continued to outperform (p= 0.006) by achieving the best rates across cleavage stages at 2-16 cell (16%; the lower value of arrest) and blastocyst (42%). Significant differences (P=0.03) were observed among the rates of Type 1embryos (the highest rate: 45.3%; Treatment F) and Type 3 embryos (the highest rate: 45.1%; Treatment A). No significant differences were observed in the rates of morula and Type 2 embryos. It is advised to add 15% of the cFF derived from follicles with a diameter of ≥3mm and 100 µg of LH ml-1 in the maturation media to obtain higher rates of maturation and cleavage of goat oocytes.

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Effect of follicle size on hormonal status of follicular fluid, oocyte ATP content, and in vitro embryo production in prepubertal sheep

Domestic Animal Endocrinology ◽

10.1016/j.domaniend.2020.106582 ◽

2021 ◽

Vol 75 ◽

pp. 106582

Author(s):

I. Contreras-Solís ◽

M. Catalá ◽

S. Soto-Heras ◽

M. Roura ◽

M.T. Paramio ◽

...

Keyword(s):

Follicular Fluid ◽

Hormonal Status ◽

Atp Content ◽

Embryo Production ◽

Follicle Size ◽

In Vitro Embryo Production

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Exposure to follicular fluid during oocyte maturation and oviductal fluid during post-maturation does not improve in vitro embryo production in the horse

Zygote ◽

10.1017/s096719941700048x ◽

2017 ◽

Vol 25 (5) ◽

pp. 612-630 ◽

Cited By ~ 4

Author(s):

Cécile Douet ◽

Olivia Parodi ◽

Nicola Antonio Martino ◽

Giovanni Michele Lacalandra ◽

Michele Nicassio ◽

...

Keyword(s):

Oocyte Maturation ◽

Follicular Fluid ◽

Culture Conditions ◽

Blastocyst Development ◽

Embryo Production ◽

Development Rates ◽

Maturation Medium ◽

In Vitro Embryo Production ◽

Oviductal Fluid

SummaryMost wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) technique for equine embryo production is available. The development of culture conditions able to mimic the maturation of the oocyte in preovulatory follicular fluid (pFF) and the post-maturation in oviductal fluid (OF) may improve embryo production in vitro. Our aim was to analyse the effect of in vitro maturation in pFF and incubation in OF on in vitro maturation of equine oocytes, fertilization using conventional IVF or ICSI, and embryo development after culture in synthetic oviductal fluid (SOF) or DMEM-F12. Oocytes collected from slaughtered mares or by ovum pick up were matured in vitro in pFF or semi-synthetic maturation medium (MM). The in vitro maturation, fertilization and development rates were not statistically different between pFF and MM. After in vitro maturation, oocytes were incubated with or without OF. Post-maturation in OF did not significantly improve the fertilization and development rates. Thus, in our study, exposure to physiological fluids for oocyte maturation and post-maturation does not improve in vitro embryo production in the horse.

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Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production

Scientific Reports ◽

10.1038/s41598-021-90810-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Francisco A. García-Vázquez ◽

Carla Moros-Nicolás ◽

Rebeca López-Úbeda ◽

Ernesto Rodríguez-Tobón ◽

Ascensión Guillén-Martínez ◽

...

Keyword(s):

Western Blot ◽

Female Genital Tract ◽

Oestrous Cycle ◽

Control Group ◽

Blastocyst Development ◽

Embryo Production ◽

Or Efficiency ◽

In Vitro Embryo Production ◽

Proteomic Analyses

AbstractRecent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

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Dietary propylene glycol and in vitro embryo production after ovum pick-up in heifers with different anti-Müllerian hormone profiles

Reproduction Fertility and Development ◽

10.1071/rd14091 ◽

2015 ◽

Vol 27 (8) ◽

pp. 1249 ◽

Cited By ~ 7

Author(s):

G. Gamarra ◽

C. Ponsart ◽

S. Lacaze ◽

B. Le Guienne ◽

P. Humblot ◽

...

Keyword(s):

Propylene Glycol ◽

Embryo Quality ◽

Control Group ◽

Follicular Growth ◽

Short Term ◽

Embryo Production ◽

Blastocyst Quality ◽

In Vitro Embryo Production ◽

Ovarian Follicular Growth

Rapid genetic improvement in cattle requires the production of high numbers of embryos of excellent quality. Increasing circulating insulin and/or glucose concentrations improves ovarian follicular growth, which may improve the response to superovulation. The measurement of anti-Müllerian hormone (AMH) can help predict an animal’s response to superovulation treatment. The aim of the present study was to investigate whether increasing circulating insulin concentrations, through propylene glycol (PG) drenches, could improve in vitro embryo production in oestrus-synchronised superovulated heifers with different AMH profiles. Holstein heifers were grouped according to pre-experimental AMH concentrations as low (L) or high (H). The PG drench increased circulating insulin and glucose concentrations and reduced β-hydroxybutyrate and urea concentrations compared with the control group. AMH was a good predictor of follicle and oocyte numbers at ovum pick-up (OPU), and of oocyte and embryo quality (AMH H > AMH L). PG in the AMH H group increased the number of follicles and blastocyst quality above that in the control group, but did not improve these parameters in the AMH L group. These results indicate that short-term oral PG supplementation modifies an animal’s metabolic milieu and is effective in improving in vitro embryo production, after superovulation–OPU, more markedly in heifers with high rather than low AMH concentrations.

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176 SERUM ANTIMULLERIAN HORMONE CONCENTRATIONS AND IN VITRO EMBRYO PRODUCTION IN BEEF CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab176 ◽

2016 ◽

Vol 28 (2) ◽

pp. 219

Author(s):

G. A. Bó ◽

I. Anduaga ◽

B. Bernal ◽

G. Moreira ◽

A. Cedeño ◽

...

Keyword(s):

Pearson Correlation ◽

Oestrous Cycle ◽

Serum Concentrations ◽

Embryo Production ◽

Antral Follicles ◽

Antimullerian Hormone ◽

In Vitro Embryo Production ◽

Antimüllerian Hormone ◽

Follicle Aspiration

The collection of high numbers of oocytes for in vitro embryo production in the cow depends on the number of antral follicles available for aspirations. The number of antral follicles varies significantly among cows and can be estimated by ovarian ultrasonography or measurement of serum concentrations of antimullerian hormone (AMH). The AMH is secreted by the granulosa cells of small antral follicles and serum concentrations are independent of the stage of the oestrous cycle. Therefore, it may be useful as a tool to select oocyte donors for in vitro embryo production. An experiment was designed to evaluate the effect of breed of cattle and the correlation of serum AMH concentrations on the number of viable oocytes and embryos produced in vitro. Cumulus-oocyte complex were collected during 44 ultrasound-guided follicle aspiration sessions performed at random stages of the oestrous cycle without superstimulation in 14 Bonsmara, 11 Braford, and 19 Brangus cows that were used for commercial in vitro embryo production using frozen-thawed semen from bulls of the same breed. Blood samples were collected by jugular venipuncture at the time of OPU, centrifuged at 3000 × g for 30 min for serum separation which was frozen at –20°C until AMH analysis. Serum AMH was evaluated using an ELISA (AnshLabs, Webster, TX, USA). The sensitivity of the AMH assay was 0.011 ng mL–1 and intra-assay CV were <5%. The number of viable oocytes collected and embryos produced in each breed were compared by ANOVA and means were compared by the protected l.s.d. test when ANOVA revealed statistically significant differences. Regression analysis and Pearson correlation were used to determine the relationship between the number of viable oocytes aspirated and the number of embryos produced. The mean (± SEM) number of viable oocytes collected was greater (P < 0.05) from Brangus (23.3 ± 4.3) and Braford (22.5 ± 3.6) than from Bonsmara donors (12.2 ± 2.5). Although not statistically different, the numbers of embryos produced followed the same pattern (Brangus, 4.4 ± 0.9; Braford, 3.4 ± 0.6; Bonsmara, 2.9 ± 0.5). Serum AMH concentration was positively correlated with the number of viable oocytes collected in all breeds (R2 = 0.63 for Bonsmara, 0.50 for Braford, 0.32 for Brangus; P < 0.01). Serum AMH concentrations were also correlated with the number of embryos produced in all breeds (R2 = 0.32 for Bonsmara, 0.16 for Braford, 0.39 for Brangus). However, AMH concentrations were not significantly correlated with the proportion of viable oocytes collected and embryos produced. Results demonstrate a breed effect on the number of oocytes collected per session and that circulating AMH concentrations are correlated with the number of viable oocytes collected per session in commercial in vitro embryo production programs.

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74 Follicular fluid anti-Müllerian hormone concentration predicts juvenile ovine in vitro embryo development

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab74 ◽

2019 ◽

Vol 31 (1) ◽

pp. 162

Author(s):

J. E. Seccafien ◽

J. M. Kelly ◽

H. McGrice ◽

D. O. Kleemann ◽

K. L. Kind ◽

...

Keyword(s):

Oocyte Maturation ◽

Follicular Fluid ◽

Individual Variability ◽

Developmental Competence ◽

Response To Treatment ◽

Blastocyst Development ◽

Embryo Production ◽

Oocyte Developmental Competence ◽

In Vitro Embryo Production

Currently, the commercial viability of assisted reproductive embryo technologies within the Australian livestock industry is restricted by individual variability in response to treatment protocols as well as oocyte developmental competence. The majority of losses come from embryo wastage, resulting from poor developmental competence during in vitro embryo production. Follicular fluid is readily available when oocytes are collected for in vitro embryo production from juvenile or mature ewes, making it an appropriate target for analysis of phenotypic markers of oocyte developmental competence. Plasma anti-Müllerian hormone (AMH) is correlated with pregnancy losses, oocyte recovery, and blastocyst development in sheep and cattle and is an indicator for donors that respond best to gonadotrophin stimulation protocols in sheep, cattle, and goats. The aim of the current work was to determine the relationship between follicular fluid AMH and in vitro embryo production outcomes in sheep. Briefly, pairs of ovaries from 38 abattoir-derived lambs were collected individually and transferred to the laboratory. Ovaries were aspirated for in vitro embryo production following previously described methods (Walker et al. 1996 Biol. Reprod. 55, 703-708) and follicles counted. Aspirated oocytes from each of the 38 individual lamb’s pair of ovaries were pooled [n=4.11±0.53 cumulus-oocyte complexes (COC) matured/lamb; total COC matured=156], and remained as such during maturation, fertilisation, and culture. The remaining follicular fluid was centrifuged for 10min at 3000 rpm to remove excess cells and frozen at −20°C. The AMH was measured in follicular fluid by a human AMH Gen II ELISA kit validated for ovine samples (A79766, Beckman Coulter, Brea, CA, USA). Correlations between follicular fluid AMH levels and oocyte maturation and blastocyst development were determined using simple linear regression. Animals were divided into groups based on AMH levels [low (0.5-10.8ng mL−1), medium (10.81-17.89ng mL−1), or high (17.9-19.25ng mL−1)], with an unbalanced ANOVA used to determine group effects on oocyte maturation and blastocyst development (GenStat 18th edition, VSN International, Hemel Hempstead, UK). Follicular fluid AMH was positively correlated (P<0.05) with the number of follicles greater than 2mm (r2=0.120) and the proportion of COC cleaved from recovered oocytes (r2=0.134). The number of COC matured per lamb was greater for those with high and medium versus low AMH (5.6±0.97 and 4.4±0.72 versus 2.1±0.97 COC/lamb). Animals with high AMH produced more blastocysts than those with medium or low AMH, when expressed as a proportion of COC recovered (P<0.002) or cleaved (P<0.009) oocytes. High AMH was also correlated with a greater number of expanded blastocysts produced from cleaved oocytes (P<0.042). The current data support previous evidence that AMH levels positively correlate to higher antral follicle counts. The correlation between AMH and components of oocyte developmental competence suggests intrafollicular AMH may indicate the best oocytes to use for an in vitro embryo production system.

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Laparoscopic Ovum Pick-Up Followed by In Vitro Embryo Production and Transfer in Assisted Breeding Programs for Ruminants

Animals ◽

10.3390/ani11010216 ◽

2021 ◽

Vol 11 (1) ◽

pp. 216

Author(s):

Hernan Baldassarre

Keyword(s):

Pregnancy Rate ◽

Commercial Application ◽

Embryo Production ◽

Breeding Programs ◽

Sheep And Goats ◽

Embryo Recovery ◽

Marker Selection ◽

Holstein Calves ◽

In Vitro Embryo Production

The potential of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) as a tool for accelerated genetic programs in ruminants is reviewed in this article. In sheep and goats, the LOPU-IVEP platform offers the possibility of producing more offspring from elite females, as the procedure is minimally invasive and can be repeated more times and more frequently in the same animals compared with conventional surgical embryo recovery. On average, ~10 and ~14 viable oocytes are recovered by LOPU from sheep and goats, respectively, which results in 3–5 transferable embryos and >50% pregnancy rate after transfer. LOPU-IVEP has also been applied to prepubertal ruminants of 2–6 months of age, including bovine and buffalo calves. In dairy cattle, the technology has gained momentum in the past few years stemming from the development of genetic marker selection that has allowed predicting the production phenotype of dairy females from shortly after birth. In Holstein calves, we obtained an average of ~22 viable oocytes and ~20% transferable blastocyst rate, followed by >50% pregnancy rate after transfer, declaring the platform ready for commercial application. The present and future of this technology are discussed with a focus on improvements and research needed.

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