Sheep Sex Preselection Using Mineral Solution Injection

PROPOSED TITLE Sex preselection of sheep using mineral solution injection via subcutaneous Dr Edrees Abdullah Hamadameen Lecturer (Reproductive Physiology) Dr Abdulqadr Ahmed Hussein- Assit. Prof. Animal nutrition Mineral Metabolism 3/19/2025 1

1. Introduction Several studies have tested the factors that influence the rate of sex constitution, including the timing of mating in mice (Krackow and Burgoyne, 1997), altering the pH of seminal fluid in rabbit (Muehleis and Long, 1976), albumin separation of sperm (Beernink et al., 1993), and altering dietary mineral fed to sow (Bolet et al., 1981), rat (Vahidi and Sheikhha, 2007), chicken (Saleh and Iriyanti, 2011), dairy cattle (Stolkowski and Lorrain, 1982), or sheep (Alhimaidi et al., 2021). Both Chandraju et al. (2012) and Oun et al. (2016) have studied mothers who fed a pre-conception diet specific in its amounts of Ca, Mg. They reported that ewes received more Ca and Mg during mating had more female offspring. Similarly, results indicated that there was a potential for skewing the sex ratio toward females when Ca and Mg was used during mating (Gharibi et al., 2023). 3/19/2025 2

Nevertheless, more studies were recommending to conduct using animal module in order to elucidate the relationship between changing minerals in the diet with regard to human sex preselection (Alhimaidi et al., 2021; Gharibi et al., 2023). In Iraq, sheep play an important role as a supplier of meat, milk, and wool. Sheep are also have been used in genetic modification studies. Thus, they were used in the current study to investigate fetal sex preselection. Therefore, administration of minerals through injection could potentially provide an alternative way of dietary mineral intake. To our knowledge, the influence of an injectable solution containing CaMg around mating of ewe on local lamb sex ratio has not been studied yet. Therefore, this study aimed to investigate the effect of mineral injection via subcutaneously injection around mating on skew offspring sex ratio toward female in ewe. 3/19/2025 3

2.Materials 2.Materials and methods and methods 2.1. Location A study was carried out in ONE OF ERBIL S PRIVATE FARM. 2.2. Animals Animals (30 ewes) from one of local breeds with similar initial body weight will be used in this study. 2.3. Treatments and Routine Ewes will be allocated by LW to one of two groups. Control ewes will be injected 25 ml/ewes sterile water. While, treated will be subcutaneously injected (25ml/ewes per day) of CaMg solution every 7 days from 28 days pre- to 7 days post-mating. The 1ml of CaMg solution constitute 500 mg of Ca borogluconate and 27.5 mg of Mg hypophosphate, which provided 38 mg of Ca and 2.55 mg of Mg. Animals will be keptin a 2 groups over a period of minimum 8 weeks. The similar diet will be offered twice a day. 3/19/2025 4

2.4. Diet All Ewes will be fed the same flushing diet twice daily (09:00 and 18:00) as total mixed ration (TMR) from 28 days before to 21 days after mating. At the end of feeding flushing diet (d 21 after mating), all ewes managed as a single group and kept in similar management and feeding condition throughout pregnancy and lambing. Animals will have free access to water 3/19/2025 5

2.5. Estrous synchronization program and pregnancy diagnosis Sixteen days after the beginning of feeding the flushing diet and injection of CaMg solution, the estrous cycle of all ewes will be synchronized using CIDRs and intra-vaginally for 12 days, followed by 350 IU intramuscular injection of eCG (Gonaser 5000 IU, Hipra, Spain) immediately after CIDR will be removed. Animals will also receive an intramuscular injection of 5 mL of a solution contained vitamin A , D3, vitamin E, and Se at the day of CIDR insertion and removal, respectively. All ewes will be mated with rams for 3 consecutive days (1 ram per 5 ewes). Pregnancy diagnosis will be performed 30 days post-mating using plasma P4 assay by an ELISA reader. 3/19/2025 6

2.6. Live Weight and condition score Determination Lambs will be weighed fortnightly (assessing animal performance) on Sundays at 11:00 using a digital weigh. Body condition score (BCS) of all ewes will be recorded at one day before the beginning of CaMg injection and introduction to flushing diet (day 0), on day of CIDR removal and 30 days post- mating. 2.7. Reproductive parameters Reproductive parameters will be as followings; Estrous response (%), Fertility rate (%), Lambing rate (%), Twining rate (%), Lamb birth weight (kg/lambed ewe), Proportion of female lambs (%), Proportion of male lambs (%) 3/19/2025 7

2.8. Blood sample collection and analysis Blood samples will be collected via jugular vein puncture at week 0, mid and final week of the study. Blood samples will be centifuged at 1000 g for 15 minutes to harvest plasma or serum. Then, samples will be frozen for subsequent blood metabolites parameters. Blood samples will be analysis for all parameters (during Day of CIDR removal, Day of estrous and mating, and Day 30 after mating) for following blood parameters; Blood metabolite such total protein, blood urea, total cholesterol, and insulin Blood plasma minerals such Ca, Ma, Na, and K, Blood hormones, including Progesterone (ng/mL), Estradiol (pg/mL), Testosterone (ng/mL) 3/19/2025 8

2.9. Statistical analysis The experiment will be a complete randomised design (CRD). Daily Live Weight Gain (DLWG) will calculated by regression analysis. One-way analysis of variance (ANOVA) will be carried out to test for significant differences in performance, reproductive, and blood metabolites parameters using Genstat version 19.1 (VSN Int. Ltd., Oxford, UK). Fisher's unprotected least significant difference test was used between the means at a significant level of p<0.05. 3/19/2025 9

2.8. Statistical analysis The experiment will be a complete randomised design (CRD). Simple test will be carried out to test significance body condition score, reproductive parameters, and blood metabolite parameters using Genstat version 19.1 (VSN Int. Ltd., Oxford, UK). 3/19/2025 10

3. References Alhimaidi A.R., Aiman A.A., Muath Q.A., Alsaiady M.Y., Amran R.A. and Swelum. A.A. (2021). Sex preselection of sheep embryo by altering the minerals of maternal nutrition. Saudi J. Biol. Sci. 28, 680-684. Bolet G., Gueguen L., Dando P. and Ollivier L. (1981). Influence of mineral diet of the sow on the sex ratio of the newborn. Reprod. Nutr. Dev. 22, 1073-1081. Chandraju S., Ashraf B. and Chidan K.C.S. (2012). Impact of calcium and magnesium ions in identification of baby gender in high-sugar hamsters. J. Pharm. Sci. Res. 3, 1619-1624. Clayton E.H., Wilkins J.F. and Friend M.A. (2016). Increased proportion of female lambs by feeding Border Leicester Merino ewes a diet high in omega-6 fatty acids around mating. Anim. Prod. Sci. 56, 824-833. Gharibi, Z., Shamsolahi, M., Fatahnia, F., Mohammadi, Y. and Shokri, A.N., 2023. Effect of Calcium and Magnesium Supplementation of Ewes during Pre-and Post-Mating on Lamb Sex Ratio. Iranian Journal of Applied Animal Science, 13(1), pp.67-75. Krackow, S. and Burgoyne, P.S., 1997. Timing of mating, developmental asynchrony and the sex ratio in mice. Physiology & behavior, 63(1), pp.81-84. Navara K.J. (2018). Choosing Sexes: Mechanisms and Adaptive Patterns of Sex Allocation in Vertebrates. Springer, Cham, Switzerland. Oun A.E., Bakry S., Soltan S., Taha A. and Kadry E. (2016). Preconceptional minerals administration skewed sex ratio in rat offspring. J. Obstet. Gynaecol. Res. 4, 11-15. Saleh D.M. and Iriyanti N. (2011). The effect of adding sodium potassium and calcium-magnesium to the diet of native chicken hen on sex ratio of native chicks. Anim. Prod. 12, 12- 15 Vahidi A.R. and Sheikhha M.H. (2007). Comparing the effects of sodium and potassium diet with calcium and magnesium diet on sex ratio of rat s offspring. Pakistan J. Nutr. 6, 44-48. 3/19/2025 11

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