Does the addition of follicular fluid in the in vitro maturation medium increase the oocyte maturation and embryo production in alpacas?

In alpacas (Vicugna pacos), the high cost of in vitro embryo production is also a consequence of the use of several substances in the culture medium. In addition, embryo production rates in this species are still considered low. Thus, in attempt to reduce the cost and to improve the in vitro embryo production rates, this study evaluates the effect of adding follicular fluid (FF) in the in vitro maturation medium on oocyte maturation and subsequent embryo production. After ovary collection at the local slaughterhouse, the oocytes were recovered, selected, and allocated in experimental groups: standard maturation medium (G1) and simplified medium added by 10% FF (G2). The FF was acquired from follicles between 7- and 12-mm diameter. The cumulus cell expansion and the embryo production rates were analyzed by chi-square with p < 0.05. No differences (p > 0.05) were observed in maturation rate between G1 (66.36%) and G2 (63.12%) groups. Likewise, no significant difference (p > 0.05) was verified between G1 and G2 for morula (40.85 vs 38.45%), blastocyst (7.01 vs 6.93%), and total number of embryos (47.87 vs 45.38%). In conclusion, it was possible to simplify the medium used for in vitro maturation of alpaca oocytes resulting in embryo production rates similar to the standard medium.

Factors affecting the in vitro embryo production in buffalo (Bubalus bubalis): A review.

Under natural and well-managed conditions, the buffalo has good reproductive and productive indices. However, in vitro embryo production (IVEP) has been used commercially to maximise the number of elite animals. In this species, several factors (donor management, in vitro culture medium, semen, in vitro conditions, embryo transfer) still affect the IVEP results. In addition, the cost of this technique is very high for this purpose. Therefore, more studies, as well as adequate plans, are needed to achieve this objective efficiently. In this review, we discussed the current commercial status, influencing factors (in vivo and in vitro), and the progress and future challenges of IVEP in buffalo. A total of 81 references were used from 1979 to 2022. The relevant data or literature were searched using the following databases: Google, ResearchGate, Science Alert, Science Direct and PubMed, using the following keywords: buffalo oocytes/COCs, buffalo embryos, pregnancy and calving or live birth rate after embryo transfer. The best maturation, cleavage and blastocyst rates in the in vitro production of buffalo embryos were 95.8, 75.2 and 33.4%, respectively. The pregnancy and live birth rates ranged from 22.2% to 43.5% and from 15.3% to 36.5%, respectively, after the transfer of fresh embryos produced in vitro to the recipients. This review will help to contextualise IVEP in buffaloes, as well as create an adequate plan for implementing IVEP in buffaloes.

Use of logistic models to evaluate the response of superovulation treatment and embryo production in Santa Inês ewes.

The study aimed to verify the influence of the FecGE mutation in superovulated ewes and to evaluate the probability of logistic models to determine the response capacity of these ewes to superovulatory treatment. Santa Inês ewes (n = 29) were genotyped for the FecGE mutation and separated for their genotype group in carriers of the mutant E allele (FecGE/E, FecG+/E) and non-carrier (FecG+/+) alleles. The ewes underwent hormonal treatment for superovulation. Aside from the genotypes, variables included in the statistical model were reproductive status (empty, early lactation, or late lactation), age (> or < 6 years), and number of births (nulliparous, primiparous, multiparous). The carriers of the mutation could be discriminated from the non-carriers based on the number of corpora lutea, rate of frozen embryos, and fecundity. Recovery rate was significantly higher (P < 0.05) in FecGE/E (94.31%) compared to FecG+/E (63.15%) and FecG+/+ (61.90%) (P < 0.05), whereas fecundity rate of FecG+/+ ewes (50.76%) was significantly higher than FecG+/E (18.96%) and FecGE/E (32.53%) (P < 0.05). We determined in this study that the response to superovulation and embryo production can be discriminated between FecGE/E and FecG+/E ewes in relation to the FecG+/+ genotype. Logistic models that included reproductive status and mutation, or reproductive status and age, or reproductive status and number of births were effective in predicting the response to superovulatory treatment.

The Rhodamine B-encrypted Vipericidin Peptide, RhoB-Ctn[1-9], Displays In vitro Antimicrobial Activity Against Opportunistic Bacteria and Yeasts.

BACKGROUND: Crotalicidin (Ctn), a snake venom cathelicidin-related antimicrobial peptide, is a 34-residue-long linear lysine-rich vipericidin obtained from the South American rattlesnake, Crotalus durissus terrificus. Ctn contains tandem repeats of nine amino acid residues (1KRFKKFFKK9 and 16KRLKKIFKK24; consensus: 1KRhKKhFKK9, h = hydrophobic amino acid) as an integral part of its structure. OBJECTIVES: The aim of this study was to evaluate the antimicrobial activity of the encrypted vipericidin nonapeptide KRFKKFFKK, designated as Ctn[1-9], and its structural analogue, rhodamine- B‒conjugated Ctn[1-9], designated as RhoB-Ctn[1-9]. METHODS: The susceptibility of representative pathogenic bacteria and yeasts to antimicrobial agents was determined using the broth microdilution minimum inhibitory concentration (MIC) method. Cytotoxicity was estimated using a hemolytic assay. The accumulation of RhoB-Ctn[1-9] in microbial cells was observed by fluorescence microscopy. The antimicrobial synergism of RhoB-Ctn[1-9] with antimicrobials was evaluated using a checkerboard analysis. RESULTS: RhoB-conjugated Ctn[1-9] displayed selective antimicrobial activity against infectious gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and pathogenic species of Candida with low hemolytic effects on human erythrocytes which were not observed with unconjugated Ctn[1-9]. RhoB-Ctn[1-9] could permeate cell membranes and accumulate intracellularly in microbial cells. RhoB-Ctn[1-9] exhibits synergistic effects when used with antibiotics or antifungal agents and reduced the MICs of the peptide and antimicrobials. CONCLUSION: These findings indicate the potential of crotalicidin-related short peptides as structural motifs for the diversification of biological functionalities. Further, they set the stage to investigate the molecular mechanisms by which chemically modified vipericidin repeats modulate cell fate.

Occurrence, morphology, and morphometry of follicles containing multiple oocytes in FecGE mutant Santa Inês ewes.

This study was conducted to characterize the morphology and morphometry of follicles containing multiple oocytes (MOFs) and determine the association with the FecGE mutation in Santa Inês ewes. Based on the genotypes, 65 ewes were characterized as being homozygous wild-type (n = 25; FecG+/+), heterozygous mutant (n = 27, FecG+/E), and homozygous mutant (n = 13, FecGE/E). The variables evaluated were follicle developmental stage, number of oocytes per follicle, morphology, and morphometry of MOFs. The FecGE mutation did not affect the frequency of MOFs (P > 0.05) (3.0 % in FecG+/+; 3.3 % in FecG+/E; and 3.5 % in FecGE/E). The greater viability (P < 0.05) of MOFs was identified in transitory stage of the FecGE/E (95.0 %) and FecG+/E (90.9 %) when compared to the FecG+/+ genotype (73.3 %). Furthermore, the morphology of transitory follicles with two oocytes was the variable and when evaluated was the most reliable determinant for predicting which ewes had an FecGE mutation. In conclusion, the FecGE mutation did not affect the frequency of MOFs. The ewes with FecGE mutation had a greater frequency of morphologically normal MOFs in the transitory stage. Furthermore, the ewes with the FecGE mutation had a greater likelihood of having MOFs containing two morphologically normal oocytes.

Intrinsic quality of goat oocytes already found denuded at collection for in vitro embryo production.

Although cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOCs) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse DOC was obtained, regardless of maturation media (complex, semidefined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. Denuded oocytes subjected to parthenogenetic activation had greater (P < 0.05) development capacity, compared with those undergoing IVF with average cleavage rate of 83% and 75%, blastocyst rate of 49% and 28%, and blastocysts in relation to the cleaved embryos of 59% and 38, respectively. In addition, the quality of embryos evaluated after vitrification/warming was similar between parthenogenetic activation and IVF. Finally, we demonstrated that the coculture of cumulus-oocyte complex (COC) with DOC increased the competence of DOC at a ratio of 1:1 and 1:9 (DOC:COC). We believe that presence of cumulus cells (CCs) is not essential to the meiotic maturation, if at the time of removal of the oocyte from follicular environment, they already acquired competence to development. However, when the oocytes still need to acquire competence, the presence of CC may significantly contribute in their developmental capacity acquisition during IVM. Thus, regardless of the source, these oocytes will require longer time in IVM, contrary to what happens in the absence of CC. In conclusion, although DOC had a lower developmental potential, especially after IVF, they were able to produce blastocysts and the coculture of DOC with COC increased this developmental capacity.

Combination of oviduct fluid and heparin to improve monospermic zygotes production during porcine in vitro fertilization.

In vivo, the oviduct provides appropriate microenvironment conditions for monospermic fertilization and early embryo development. In addition, glycosaminoglycans such as heparin are present in the oviduct and have been shown to modulate the activity of oviduct-secreted proteins on the regulation of sperms parameters. Thus, the present study was designed to evaluate the effect of porcine oocytes exposure to oviduct fluid (OF) before in vitro fertilization (IVF; incubation of oocytes in OF for 30 minutes before IVF), during IVF (supplementation of IVF medium with 10% OF), and during IVF in combination with heparin (10% OF + 10-µg/mL heparin) on IVF parameters. Regardless of sperm concentration used (0.5, 1.5, or 4.5 × 10(5) cells/mL), exposure of oocytes to OF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control group. This resulted in a general increase (P < 0.05) in the final output of the IVF system in terms of zygotes with two pronuclei in OF-exposed groups: 56 ± 9% (OF before) and 60 ± 7% (10% OF during IVF), compared with control (21 ± 8%), when IVF was performed with 4.5 × 10(5) cells/mL. The combination of 10% OF with heparin during IVF induced a decrease (P < 0.05) of the penetration rate, with no effect (P > 0.05) on the monospermy rate in comparison with 10% OF alone. This resulted in a general reduction (P < 0.05) in the final output of the IVF system (%), which was 33 ± 6% and 52 ± 8%, for 10% OF + heparin and 10% OF, respectively. In conclusion, the OF, used in porcine IVF, exerted a beneficial effect on oocytes by reducing the incidence of polyspermy without decreasing the penetration rate. However, the association of the OF with heparin reduced the efficiency of monospermic zygotes' production.

Ovarian follicular response to different hormonal stimulation treatments in Canindé goats.

The objective of the present study was to evaluate the effect of different hormonal stimulation treatments on the antral follicular population of naturalized Canindé goats. Adult goats (n=17) having estrous cycles at regular intervals were treated with intra-vaginal sponges containing 60 mg medroxyprogesterone acetate for 11 days, combined with an application of 50 µg d-cloprostenol on the Day 8 of treatment. For ovarian stimulation, goats were distributed into the following experimental groups: (a) multiple doses (MD), with a total of 120 mg NIH-FSH P1 in five intramuscular injections (30/30; 20/20 and 20 mg) at 12-h intervals; (b) three doses (TD), with a total of 120 mg NIH-FSH P1 in three intramuscular injections (60; 40 and 20 mg) at 24 h intervals; (c) one dose (OD), which consisted of the use of 70 mg NIH-FSH P1 combined with 200 IU eCG administered intramuscularly 36 h before sponge removal. In the MD and TD groups, FSH injections were begun on the Day 8 of progestagen treatment. The ovaries of all animals were observed by transrectal real time ultrasonography (TRU) during the follicular stimulation protocols. All follicles ≥2 mm were counted, measured and classified according to greatest diameter. The ultrasonographic assessment of the ovaries provided for well-defined ovarian structures. At the time of insertion of the sponges (Day 0), significant differences were observed (P<0.05) for the mean number of large follicles between the treated groups. Meanwhile, on Day 11, the three treatments did not differ (P<0.05), regardless of the follicular category. The diameter of small follicles was similar in MD, TD and OD during the whole period of the study. In the TD group, diameter of the large follicles was less (P<0.05) on Day 10 when compared to MD and OD. However, these differences were not observed on Day 11. In conclusion, the three treatments produced comparable distribution of the follicular populations. However, the single dose treatment can be preferred because of its simplicity and efficacious follicular response.