Genetic and environmental factors have been reported to affect ovary characteristics. In the present study, we investigated the effects of metabolic profile on follicular population, oocyte quality and yield in three tropical cattle breeds. Blood and ovaries were collected from 195 zebu cattle (Gudali, Red and White Fulani) aged 3 to 15 years, with body condition score (BCS) of 2.93 ±0.65 (1 to 5) during slaughtering. The phases of sexual cycles were divided into proestrus, estrus, metestrus and diestrus. In each ovary, the follicle were counted and classified according to their diameter in small (8 mm) using an electronic caliper. Oocytes were collected by slicing technique and classified in class I, II, III and IV according to their cumulus investment and cytoplasmic distribution in four groups under a light microscope (400X). The metabolic or biochemical parameters were evaluate in the serum using automate and commercially available kits. Results show that from 390 ovaries, 6642 follicles were counted. The mean number of follicles per ovary was 18.45±0.58 and 15.74±0.48 respectively for right and left ovary. Small, medium and large follicles per cow represented 24.15±0.86; 9.30±0.39 and 0.61±0.05 respectively. The average number of follicles obtained per cow (34.06 ± 13.01) increased significantly with BCS (P<0.05). With the exception of total of class III oocytes, the overall number of follicles and oocytes were significant higher during rainy than dry season (P<0.05). Ovaries with corpus luteum presented fewer follicles than ovaries without corpus luteum. The highest number of large follicles was obtained during estrus phase of sexual cycle and the number of class I and II oocyte were obtain in proestrus. The mean oocyte yield per ovary was 8.34±0.28 and 7.02±0.24 respectively for right and left ovary. The quality of oocyte graded I, II, III and IV were 5.94 ±0.19, 3.29±0.16, 2.56 ±0.12 and 3.83±1.14 respectively. The average number of selected oocyte for in vitro embryo production represented 9.23±0.35 per cow and the oocyte index was 0.93. At the end of the research, we can easily see that, dry season negatively affect follicular population, oocyte yield and metabolic parameters. Estrus and proestrus are the best phases of the sexual cycle for the emergence of large follicles and good quality oocytes respectively.

Azafack DK, Ngoula F, Kouamo J, Tchoffo H, Kenne LK (2020) Effects of Metabolic Profile, Season and Stage of the Sexual Cycle on Follicular Dynamics, Quality and Oocyte Yield In Three Tropical Zebu Cattles. J Vet Med Res 7(2): 1185.

•    Metabolic profile
•    Sexual cycle
•    Follicular dynamics
•    Oocyte yield
•    Tropical zebu cattles 

Cattle production plays an important role in Cameroon’s national animal systems and represents 10% of total livestock [1]. Reproductive performance is vital to perpetuate existence of cattle. This highly depends on adequate nutrition and proper management system of rearing the livestock, the lack of which associated with pathological factors results in poor productivity, infertility and sometimes sterility [2]. These factors may be classified either into specific diseases affecting the reproductive organs directly or non-specific factors that underscore the reproductive efficiency [3]. Cattle breeds in Cameroon are trypano-tolerent cattle (Namchi, Kapsiki, Kuri and Bakossi) which are very robust [4] and are considered to be highly endangered breeds [5]. Nevertheless, there are principally trypano-susceptible cattle in Cameroon: zebus (Bos indicus) [6]. Despite the rich breed diversity of local cattle; their productivity still remains low due mainly to farming systems commonly extensive without steady animals. Breeding of local cattle is naturally under traditional management systems with very little or no breeding programs.

One of the most important particularities of the extensive breeding systems is exploitation of fodder resources, often heterogeneous in space and variable with time. Thus, seasonal fluctuations of food resources lead livestock to practice a dynamic of storage and mobilization of tangible reservation [7]. Evolution weight of animals enable to identify critical period and factors that limit their productivity [8].That can be studied through Body Score Conditions during different seasons, and /or metabolic profile analysis. To overcome these problems, Assisted Reproductive Technologies (ARTs) such as Artificial Insemination (AI), In Vitro Embryo Production (IVEP), multiple ovulation and embryo transfer (MOET) have been developed. In Cameroon, artificial insemination (AI) of local breeds with exotic semen has contributed to the improvement of cattle genetic potential [9]. However, embryo transfers (IT) and In Vitro Embryo Production (IVEP) of cattle has not been sufficiently tested in Cameroon. In cattle, the IVEP is largely used in the world and has allowed the production of high genetic embryos from oocytes collected from live cows or after slaughter to promote the growth of the specific species [10]. The processes of collection, maturation, fertilization and in vitro culture of oocytes were significantly improved [11] and were also used in the development of laboratory in mass production of embryos in vitro. However, the egg donor intrinsic and extrinsic factors such as follicular size, morphology of cumulus complex, biochemical, hormonal and nutritional aspects have been reported as the cause of results fluctuations in the laboratories [12]. In addition, hormones such as Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) for in vitro maturation (IVM) medium is important even in the subsequent in vitro fertilization (IVF) and embryonic development [13].

It is in this context that this study was undertaken with the main objective to evaluate factors that affect metabolic and hormonal profile of three cattle breeds in Cameroon (Gudali, White and Red Fulani). This was achieved by the determination of follicular population, oocytes quality and establishment of the relation between the reproductive characteristics of cows, the stage of the estrus cycle and season, the nutritional status of animals, the impact of energy, protein parameters and hormone on the follicular population, quality and oocyte yield.

Study area

The study was carried out in Bafoussam, west region of Cameroon (Latitude, 5°46’ N and Longitude, 10°40’E) characterized by Sudano Guinean climate. Average annual precipitation 2000 mm of water with a short dry season of 3-4 months (November to February) and long rainy season of 8-9 months (March - October). The samples were collected at the Bafoussam municipal slaughterhouse from November 2017 to October 2018 and analyzed in the Animal Physiology and Health Laboratory of the University of Dschang.

Animal selection

The study was conducted on a total of 195 cyclic local zebus of different breeds: Gudali (64), White (63) and Red Fulani (68). Cows were mainly originated from North West region and were randomly selected. Body Condition Score (BCS) and age of cows were determined as described by Natumyana et al. [14] and Moussa Garba et al. [15] respectively.

Blood collection

Blood sample was collected from the jugular vein of each cow immediately after slaughter in 10 ml dry tubes (reference: Venoject R). After collecting, blood samples were transferred to the laboratory at 5°C and centrifuged at 2700 g for 15min and the sera obtained were stored in aliquots (1.5 ml Eppendorf tubes) at -20°C until assayed.

Ovary collection and handling

After slaughter, the left and right ovaries were excised and placed in separate conical tubes containing physiological normal saline solution (0.9%) supplemented with antibiotics (0.5 mg / ml of penicillin-streptomycin sulfate). Thereafter all collections were transported immediately to the laboratory in a sterile thermos with temperature between 34 to 36°C.

Determination of weight and the size of ovary

In the laboratory, all ovaries were cleared off attached tissue and mesovarium (trimming) and weighed using an electronic scale, Mettler PC 2000. The length, width and thickness of the ovary were measured using digital calipers and the ovaries were thereafter classified into two groups in function of their volume (V < 2.25 x 1.75 x 1.25 and V> 2.25 x 1.75 x 1.25) with V= volume, 2.25 = length, 1.75 = width and 1.25 = diameter of ovary as described by Samad and Raza [16].

Determination of follicular population and stage of sexual cycle

The trimmed ovaries were subjected to washings (5-6 times) with warm saline fortified with antibiotics and transferred into the laminar flow. The apparent follicles on each ovary were measured using electronic digital calipers, counted and then classified according to their diameters (Φ) into three categories: small (Φ < 3 mm), medium (3 ≤ Φ ≤ 8 mm) and large (Φ > 8 mm) as described by Baki et al. [17]. The size and color of corpus luteum present on the ovary were noted and permitted to distinguish four stages of the sexual cycle (proestrus, estrus, metestrus and diestrus) as described by Nguyen-kien and Hanzen [18] and Satrapa et al [19].

Oocytes recovery

Oocytes were retrieved using slicing technique [20] in the separate plastic Petri dishes containing collection medium (physiological normal saline solution 0.9%). The collected oocytes were graded as excellent (I), good (II), fair (III) and poor (IV) quality under the stereo microscope (x 10) according to the homogeneity of the cytoplasm and layers cumulus of cell [21]. The index (overall quality) was calculated using the following formula [(GI x 1 + GII x 2 + GIII x 3 + GIV x 4) / Total number of oocytes recovered] when GI, GII, GII and GIV represented grate I, grate II, grate II and grate IV oocyte as described by Duygu et al. [22]. Index values that trend to one reflected good quality oocytes.

Biochemical analysis

Serum sample was analysed to determine the concentration of total cholesterol, urea, phosphorus, total protein, and albumin. Total globulins were calculated by subtracting albumin from total protein [23]. All biochemical parameters were determined by colometric methods using an Automated chemistry analyzer (Lab Max Plenno, Labtest, Lagoa Santa, Brazil. While the levels of different hormones (FSH, LH and estradiol) in the sera were determined using the commercial kits ELISA (OMEGA Diagnostic Automation Inc).

Statistical analysis

The collected data were analysed using SPSS version 20.00. Analysis of Variance 1 and 2 ways and Duncan’s test statistics were used to test the differences between means. The level of significance was recorded at the 5% level of confidence.

Characterization of slaughtered cows

The average BCS of cows were 2.93 ±0.65 on a ladder 1 to 5. The minimum and maximum BCS registered was 2 to 4. The percentages of animals with BCS equal to [3] and age equal to [6- 9] were high as well as in Gudali, Red and White Fulani (Table 1).

Follicular population

From 195 pairs of ovaries collected, 6642 follicles were counted on their surface. The mean number of follicles per ovary was 18.45 ± 0.58 and 15.74 ± 0.48 respectively for right and left ovary. Small (Φ ? 3mm), medium (3 ≤ Φ ≤ 8 mm) and large (Φ> 8 mm) follicles per cow represented 24.15 ± 0.86; 9.30 ± 0.39 and 0.61 ± 0.05 respectively. The average number of follicles obtained in this study per cow (34.06± 13.01) increased significantly (P < 0.05) with BCS (Table 6). The overall number of follicles was significant (P < 0.05) high during rainy than dry season (Table 2). In additionally, the number of large follicles and god quality oocyte were significantly (P < 0.05) high during estrus stage of sexual cycle (Table 3, 5a and 5b)

Nutritional status of cows

Metabolic dosage in 195 cow’s serum used in this study was classified into protein parameters (total protein, albumin, globulin and urea), energy (total cholesterol) and hormones (Follicle Stimulating Hormone, Luteinising Hormone and estriol). It showed that the levels of albumin and cholesterol were significantly higher in Gudali cows with BCS = 3 while the level of urea was significantly higher in thin Gudali (P < 0.05) (Table 4).

Oocyte quality

Of the total of 390 ovaries, 3045 oocyte were obtained. The mean oocyte yield per ovary was 8.34± 0.28 and 7.02 ± 0.24 respectively for right and left ovary. The oocyte quality graded I, II, III and IV (Figure 1) were 5.94 ±0.19, 3.29 ±0.16, 2.56 ±0.12 and 3.83 ± 1.14 respectively. Selected oocyte for IVEP represented 9.23 per cow and the oocyte index was 0.93. It is also shown that, with the exception of total of class III oocyte, the distribution of oocytes was significantly (P < 0.05) high during rainy than dry season. Right ovaries were heavier than left and the total number of follicle and oocyte of right ovary was higher than the sum of the left. In addition, dry season was negatively impacted ovary characteristics (Table 7a and b).

The quality and the number of oocyte were generally most important during rainy season than what reported in dry season (Table 8a and 8b).

From the following results, some correlations have been retains. It follows up highly positive correlations between the BCS and the average ovary weight (r = 0,28** ; P < 0,01), between the total number of follicle and the total number of small follicle (r = 0,99** ; P < 0,01) ; between the rate of total protein and class I oocyte (r = 0,15* ; P < 0,05). Nevertheless, highly negative correlations have been recorded on the one hand between the total number of large and medium follicle (r = -0,19** ; P < 0,01), on the other hand between the total number on large follicle and class III (r = -0,15* ; P < 0,05) (Tables 9-11).

Reproductive efficiency as well as Body Condition score may be affected by a season as observed in this study. The lowest BCS of animal obtained in dry season may be due on the one hand to lack of feeds or feeds supply with poor nutriments but no doubt related to bad healthy status associated to most important development of epizootic diseases and some infections in dry season and also on the other hand, to the availability of feeds during the dry season. In fact, Maina et al. [24] has demonstrated the under nutrition of Africa zebu cows. In fact, cow with BCS ≤ 2 have a fewer developing follicles during the luteal phase of the estrous cycle and tend to produce fewer eggs during the follicular phase compared to those with BCS ≥ 3 because the number of follicles that leave the ovarian reserve depends of the individuals BCS [25]. Generally, periods of low nutrition are associated with a decrease in insulin secretion and elevation of nonesterified fatty acids (NEFA) [26] due to enhanced lipolysis and reduced lipogenesis. Nevertheless the lowest BCS of cows was generally less sensitive on younger than older cows. This may be due to age as reported by Ali et al. [27]. In fact, appreciable growth weight of young animals attenuated harmful effects of unfavorable season and then they are more resistant during strong conditions of dry season [28].

The average number of follicles obtained in this study per cow increased with BCS, also in rainy season inside different breeds; confirmed the fact that energizing supply has a major impact on dynamic of follicular growth [29]. The higher number of follicles and oocyte accounted on the ovary in the rainy season was reported by Diskin et al. [30] and Fassi et al. [31] who indicated that ovarian activity is most important during rainy season. This may be attributed to availability and quality of pasture in rainy season which enhance chance of feeds. Thus, they confirmed that the nutritional status of the animal had an influence on the ovarian function in zebu cows, likewise the season of the year [32]. The metabolic and hormonal changes between the rainy and dry season affect follicular dynamics and oocyte development. This may be due to heat stress which altered endocrine patterns and reduced follicular development. In fact, heat stress induces the production of heat-shock proteins which may serve as protection against thermal stress [33]. Then, these proteins could constitute the lack of proteins used in follicular growth and oocytes development. In addition, heat temperature increases the number of small follicles as well as observed in this study and oocytes are in bad quality [34]. The higher degeneration of oocytes in dry season reflects their lower developmental competence compared with oocytes recovered in rainy season. Thus, during oogenesis and folliculogenesis, several dynamic processes that are regulated by endocrine, paracrine and autocrine signals have been shown to be linked with energetic status and the quality of oocyte depends on the nutritional condition under which the follicle began its development [35]. Moreover, oocyte growth and maturation are believed to be particularly sensitive to changes in nutritional, chemical and endocrine environments.

Significantly higher follicles were observed in this study during estrus stage of sexual cycle. This result is in accordance with the findings of Kouamo et al. [36] using zebu cows in Adamawa region of Cameroon. In fact, in estrus stage, the follicles are large in size and only one becomes dominat (12-13 mm) while the others are atresia [37]. This might explain the reason which more large follicles were counted in this stage. Mutha and Uma [38] reported that the oocyte yield and the quality of cultivable oocytes are higher when the cows are in the follicular (proestrus and estrus) than the luteal (metestrus and diestrus) stage. Thus follicular stage is the best period of the sexual cycle for the emergence of quality oocytes.

The right ovary was heavier than the left suggesting that right ovary is more active than the left. The same observations were reported by Kouamo et al. [36]. Indeed, the finding of Drion et al. [39] showed that there exist in cattle a great follicular activity on right ovary and consequently ovulations are more regular at this level. In addition, a density of blood vessels which facilitates blood supply are must important on right ovary than left.

The not significantly higher level of protein, but significantly higher level of urea and phosphorus obtained in Gudali cow in this study could be attributed to moderate BCS observed with this breed. Indeed, protein provides amino acids that are necessary for maintenance of vital functions as growth, reproduction and lactation, while serum urea is an indicator of the balance between nitrogen and energy intake. Whereas phosphorus deficiency act especially at the ovarian level by anoestrus, irregular estrus, and decreasing of ovarian activity [40]. This substantiated the previous results of follicles and oocyte yield obtained with this breed.

Low concentrations of hormones obtained in this study during dry season are attributed to the bad and lack of feeding due to the characteristics of strong season. This agrees with earlier findings of Ali et al. [27]. Indeed, periods of low nutrition are generally associated with a decrease in insulin growth factor (IGF) secretion and elevation of nonesterified fatty acid (NEFA) [26]; and then may cause the lengthening of the sex cycle durations in zebus. It has been proven that, insulin may serve as a nutrition signal influencing LH release [41]. On the other hand, it has been reported that, energy balance, specific hormones and metabolites remain precise indicator [42]. In addition, nutrition related metabolic and hormonal changes may affect both follicular growth and oocyte quality yield [43]. In fact, cows with negative energy balance may have direct consequences on the hypothalamic- pituitary axis by reducing the secretion of pituitary gonadotropins especially FSH and LH which are responsible for follicular development and oocyte mature [29]. Inversely, high plasma levels of IGF-1 resulting from improved nutrition, increases the sensitivity of granulosa cells to FSH stimulation [44]. Indeed, basal folliculogenesis is essentially controlled by growth hormones such as Insulin Growth Factor-1 (IGF-1) and the lack of which could be resulting poor follicular yield and oocyte quality of cows.

This study indicated that season temperature had a detrimental effect on the follicular population, oocyte yield and metabolic parameters. Moreover for the IVEP, females’ oocyte donors should have acceptable BCS and oocyte should be collected during proestrus and estrus stage of sexual cycle of cows.

The authors gratefully acknowledge to the head of the zootechnical center of Bafoussam, the butchers of the Bafoussam municipal slaughterhouse for making available animals for the research facilities of contribution

Experimental protocols used in this study were approved by the ethic comity of the Department of Animal science, FASA, University of Dschang - Cameroon and strictly conformed with the internationally accepted standard ethical guidelines for laboratory animal use and care as described in the European Community guidelines; EEC Directive 86/609/EEC, of the 24th November 1986.

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