Oocyte quality in adapted Bos taurus taurus cows

Abstract The objective of the current study was to determine the performance of creole cows of the Chino Santandereano breed in the ultrasound-guided follicular aspiration technique (OPU) and the quality of oocytes recovered. A total of 15 multiparous cows were selected from a herd located in the department of Santander with tropical climate. The cows were submitted to 5 sessions of follicular aspiration with an interval of 45 days. In each aspiration session, 7.9 ± 0.7 oocytes were recovered per cow, with a recovery rate of 64.9% ± 0.5 and a rate of viable and non-viable oocytes of 64.3% ± 0.6 and 35.7% ± 0.7, respectively. A difference (P < 0.05) in oocyte quality was presented for grades I, II and III (7.7 ± 0.4, 19.8 ± 0.5 and 36.8 ± 0.5, respectively). The results suggest that Chino Santandereano cows submitted to the OPU technique have a higher percentage of grade III oocytes recovered.


Introduction
Exposure to a process of natural selection over several generations led Colombian creole cattle to adapt to a wide variety of local conditions, developing characteristics of survival to feed shortage, continuous productive and reproductive performance even during periods of stress, 1 high resistance to diseases and/or parasites, high tolerance to adverse climatic conditions and greater longevity. 2raditional livestock systems in Colombia are characterized using locally adapted cattle breeds (Bos taurus taurus) and their crosses, however, with the industrialization of livestock farming, the entry of improved breeds was carried out, which gradually displaced the native breeds, currently affecting an important animal genetic resource such as the Chino Santandereano breed, catalogued today in danger of disappearing. 3n Colombia, there are very few reports related to the study of the reproductive physiology of creole breeds and the performance of these breeds during the application of different assisted reproduction techniques.The OPU technique in this important zoogenetic resource can be of great utility for the conservation and preservation of said species, however, it is widely known that this technique suffers extreme individual variation due to biological factors such as breed or genetic group, 4 age, 5 reproductive status, 6 estrous cycle phase 7 and metabolic status. 8ny exogenous factors with negative influences or intrinsic oocyte abnormality will compromise the fertilizing potential of oocytes. 9As a prerequisite to obtain a healthy embryo is first obtaining a healthy oocyte.Thus, improved access to maternally regulated factors will allow a better understanding of the mechanisms and pathways regulating gametogenesis and embryogenesis that can possibly alleviate the reduced cattle reproductive performance observed worldwide and may facilitate discovery of novel contributors to early embryonic development with the ultimate goal of improving oocyte quality. 10 study carried out in Gyr breed cows (Bos taurus indicus) recovered 4.4 to 7 oocytes per aspiration, with a percentage of grade I structures of 8 to 13.6% and a rate of viable oocytes between 40 to 63.6%. 11ontes et al. 12 and Gonz alez 13,14 in the same genetic group obtained an average of 30 oocytes per aspiration session per donor, with a viable oocyte rate of 77.6%.Works performed in Bos taurus taurus females [15][16][17] presented oocyte recovery rates from 37 to 72.2%, with a few structures recovered from 2.6 to 8.2.
For this reason, the current study aims to evaluate for the first time the efficiency of the follicular aspiration technique and the quality of oocytes recovered from creole cows of the Chino Santandereano breed.

Material and methods
The present study was developed out in accordance with the laws and regulations of Colombia, established by resolution 8430 of 1993, which provides for biomedical research with animals.The study was approved by the Ethics and Bioethics Committee of the University of Santander, minute No. 18 of June 11, 2019.

Selection of the animals
Fifteen multiparous cows of the Chino Santandereano breed (adapted Bos taurus taurus), non-lactating, nonpregnant, aged between 3 and 7 years, with a body condition status of 3.0 ± 0.4 on a scale of 1 to 5 points and with normal reproductive tract at a structural and functional level, were used.The cows were selected from a nucleus located in the department of Santander (Colombia), free of brucellosis and tuberculosis, and vaccinated against foot and mouth disease.The study was carried out under tropical conditions with an average temperature during the whole experimental period of 39 C, with pastures based on Brachiaria decumbens, mineral supplementation and water ad libitum.The handling of the animals was carried out in cattle handling pens, a facility that allows safe containment and preservation of animal welfare.

Experimental design
Each animal was considered as an experimental unit and a total of five OPU sessions were performed, with an interval of 45 days between aspirations.

Ultrasonography-guided follicular aspiration
The OPU was performed on a random day of the estrous cycle on those follicles with a diameter 3 mm and were classified into three groups: small follicles (5 mm), medium follicles (5 to 9 mm) and large follicles (9 mm).Before OPU was performed sedation of the animals with 2% xylazine (0.02 mg/kg) and the application of epidural anesthesia between the last sacral vertebra and the first coccygeal vertebra by administering 0.2 mg/kg lidocaine (Roncaina V R , Ropsohnlab, Colombia).Follicles with a diameter 3 mm to 9 mm were aspirated, OPU was performed with the aid of a disposable 18 G x 75 mm needle, coupled to a Teflon line with a negative pressure of 70 mm/Hg.Prior to the aspiration procedure, the follicles in both ovaries were counted using an ultrasound scanner (Mindray, DP 2200 VET, China), equipped with a 7.5 MHz microconvex transducer, coupled to a transvaginal guide for follicular aspiration.
A 50 mL conical tube was used for each animal, containing a volume of 5 mL of D-PBS (D-PBS V R , Vitrocell, Brazil), supplemented with 0.1% heparin (Liquemine V R , Roche, Brazil) and 1% fetal bovine serum (Gibco BRL, Grand Island, NY).Immediately after puncture, the follicular fluid was transferred to an EmCon filter (Agtech, USA) and 100 mL of D-PBS was added to remove clots and cells.The structures were washed in commercial HEPES-buffered medium (Wash, IVF Bioscience, UK).
COCs were classified based on the number of cumulus cell layers and morphological appearance. 18OCs with more than three compact layers of cumulus cells were classified as grade 1 (G1); COCs with at least one layer of cumulus cells and oocytes with slightly heterogeneous cytoplasm as grade 2 (G2); partially nude COCs and oocytes with heterogeneous cytoplasm as grade 3 (G3).Were included in the calculation of the recovery rate and number of total oocytes.

Statistical analysis
The data were analyzed using Statgraphics Centurion XVIII software.Means and standard deviation were obtained, being analyzed by ANOVA the number of follicles visualized, number of oocytes recovered, recovery rate, percentage of oocytes recovered grade I, II and III and rate of viable oocytes.In the cases in which a significant effect was obtained (P < 0.05), they were compared by Fisher's LSD method.
In cattle the follicular population is considered a highly variable characteristic, 19 with a range of 8 to 56 structures visualized, thus, being considered a characteristic of high repeatability (0.84-0.95). 20,21Studies evidenced that the antral follicular population is directly correlated to the genetic group, being females of Bos taurus indicus breeds the ones that present higher quantity of antral follicles than Bos taurus taurus females. 14,22,23Several studies have classified bovine follicular population into 3 categories: low follicular count (LCF) (15), intermediate follicular count (ICF) (16 to 24) and high follicular count (HCF) (25). 21,24,25According to this information, the Chino Santandereano cows in the current study can be categorized as low follicular count.A study conducted on Podolic cows (Bos taurus tauruscreole Ukrainian breed) showed a reduced number of follicles per donor (5.5 structures). 26According to Silva-Santos et al., 27 the low follicular count of taurine breeds is related to the high rates of follicular atresia present during pre-antral follicular development.Similar results were obtained by Gonz alez, 13 in Sanmartineras creole cows, which presented an antral follicular population of 11.8 structures.Meanwhile, Narv aez and Silva 16 reported an average of 30 follicles per wave of follicular growth during the estrous cycle in creole cows of the Blanco Orejinegro breed.These results indicate that the antral follicular population of Colombian Criollo breeds may be a reproductive characteristic of variable expression.Ireland et al. 28 observed that the enzyme related to cell apoptosis (Cathepsin) was increased 2 to 6 times more in cumulus cells of LCF females relative to HCF females.In addition, they identified that the number of healthy oocytes was higher for HCF cows compared to LCF cows.
Although circulating levels of anti-M€ ullerian hormone were not quantified in the present study.Previous study showed a high correlation between antral follicular population and anti-M€ ullerian hormone concentration in Bos taurus indicus heifers. 29lward and Bohlen 30 determined that low concentrations of this hormone are present in animals with low number of antral follicles.Another mechanism that has been related to HCF is insulin and insulin-like growth factor type 1 (IGF-1), being observed that the higher the plasma concentration of these hormones, the higher the number of antral follicles, mainly obtained in Bos taurus indicus females. 31n the current study, a higher percentage of grade III oocytes was recovered in relation to grades I and II.Similar results were reported by Alvarado et al., 32 in creole cows from the Ecuadorian Altiplano, where the frequency of grade I, II and III oocytes were 22.1%, 33.6% and 44.3%, respectively.Presicce et al. 26 recovered a low number of grades I, II and III oocytes in creole Podolic cows.Few studies have been developed evaluating oocyte quality in bovine breeds adapted to the tropics, however, we consider that the high temperatures (39 C) presented during the experimental period could have affected the density of cumulus cells recovered during OPU.
The best oocyte quality presented in in vitro embryo production programs is in females of zebu breeds, a condition that is related to higher circulating levels of IGF-1 and insulin.According to Mingotti, 33 an alteration of the metabolic profile due to unfavorable nutritional conditions can reduce oocyte quality and affect in vitro embryo production programs.According to this information, further studies are needed to clarify if the low oocyte quality is a particularity of the Chino Santandereano or if it is the effect of some biological factors.The understanding of this mechanism is crucial for the development of specific reproductive strategies, which will allow the design of conservation and preservation programs for breeds in vulnerable conditions.

Conclusions
In conclusion, our results indicate that Creole cows of the Chino Santandereano breed subjected to the OPU technique present a reduced efficiency in terms of