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HORMONAL INDUCTION AND SYNCHRONIZATION OF ESTRUS IN MICE
H.O.M. Alhilfi1*, E.S. Ahmed2
1 Dep. of Environmental Biotechnology, Biotechnology Research Center, Al-Nahrain University, Jadriyah, Baghdad, Iraq;
2 Dep. of Biomedical Engineering, College of Engineering, Al-Nahrain University, Jadriyah, Baghdad, Iraq. * Corresponding author: [email protected]
Abstract. The present study was carried out to evaluate different estrus synchronization protocols in mice and follow up the proportion of delivery and gender of litters. Five protocols were tested with total number of 48 adult females that divided equally into six groups including control. Total number of 24 adult males were divided equally and introduced for mating for four days. Hormonal synchronization including intraperitoneal administration of single or double dose of 0.5 ^g Cloprostenol (Prostaglandin F(PGF)) and 3 ^g of Progesterone (P4), with or without 5 IU of equine Chorionic Gonadotropin (eCG) in five protocols. Results showed that estrus and mating rate increase significantly after administration of PGF (P < 0.05). However, low delivery rate was evident in all groups. There were no differences in the average number and the proportion of gender of litters within groups. In conclusion, synchronization of estrus in mice was not fully achieved using the current protocols. However, administration of prostaglandin increases mating rate, but the pregnancy success might fundamentally depend on other factors such as managemental.
Keywords: synchronization, estrus, mating, conception, pregnancy, prostaglandin, progesterone, mice.
List of Abbreviations
PGF - Prostaglandin F P4 - Progesterone
eCG - Equine chorionic gonadotropin ES - Estrus synchronization FSH - Follicle stimulating hormone GnRH - Gonadotropin releasing hormone NFB - Negative feedback mechanism LH - Luteinizing hormone MUNPs - Major Urinary Proteins VNO - Vomeronasal organ MOB - Major olfactory bulb
Introduction
Estrus synchronization (ES) is a practice that widely used to adjust breeding and delivery time in animal. And the detection of estrus plays the key role in the determination of suc-cessfulness of estrus synchrony. Whereas, male and whitten effect are usual practice to induce estrus through exposure of female to male pher-omones in mice (Zakaria & Sukardi, 2019), several studies reported that hormonal therapy is a common practice to induce or synchronize estrus in domesticated animal species such as bovine (Jeong et al., 2013; Frares et al., 2013 and Ahlawat et al., 2015), ovine (Gonzalez-Bulnes et al., 2020; Yu et al., 2022), caprine
(Omontese BO, 2018; Skliarov et al., 2021), and swine (Knox RV, 2015; Mavromati & Turmalaj, 2021).
There were numerous physiological factors that might determine subsequent fertilization and pregnancy rate following estrus synchronization. In bovine these mainly include nutrition, good body condition and health, male semen, and efficient estrus detection technique (Smith et al., 2013). In mice, short estrus cycle (Approximately 4-5 days) and short estrus phase (12_48 hrs) were the main challenge that made few researchers to investigate estrus synchrony in these animals (Wei et al., 2015; Ajayi & Akhigbe, 2020).
The administration of Prostaglandin F2 or its analogue D-Cloprostenol is widely used in practice for lysis of the corpus luteum and induction of estrus in most animal species including mice (Zhang et al., 2013; Wen et al., 2020). Moreover, the hormonal administration of Equine chorionic gonadotropin (eCG), which had mainly Follicle Stimulating Hormone (FSH) effect, is commonly used in superovulation regimes (Crispo et al., 2021; Shindo et al., 2022). Whereas few studies reported that eCG or PMSG reduces the viability and weight of newborns, it increases number of mice that were in
estrus (Zhang et al, 2021; Sari et al., 2022). On the other hand, administration of progesterone had suppressive effect on pulse frequency secretion of gonadotropin releasing hormone (GnRH). This effect if removed, the female will display estrus due to the increase of GnRH pulse and pituitary gonadotropins as well known negative feedback mechanism (NFB) (Herbison AE., 2020).
The Morphological evaluation of external genitalia and or cytological examination of vaginal smears were utilized to determine estrus phase in mice. However, it was reported that the degree of invasiveness when performing vaginal swaps in mice might stimulate the inflammatory response and disrupt vaginal cytology (Gal et al., 2014). Also, several factors might play a vital role in determination of copulation outcome and pregnancy in mice. Few studies reported that the proportion of mice that show estrus is exceeding 60% after estrus synchronization regimes (Sari et al., 2022; Hasegawa et al., 2017). However, scarce evidences reporting the proportion of delivered dams and number of litters as a result of estrus synchronization in mice. The current study was performed to determine the effectiveness of some synchronization protocols and follow-up delivery of dams and gender of litters in mice.
Material and Methods
Chemicals
Synthetic prostaglandin F23 (D-Clo-prostenol sodium, Galapán TM, 75 |ig per ml) was purchased from Invesa industrial veterinaria S.A. (Barcelona, Spain). Equine chorionic gonadotropin (eCG, Novormon TM 5000, 200 IU per ml) was purchased from Syntex S.A. (Argentina) and prepared according to manufacturer instructions. Progesterone (Vetagester-one ®, 25 mg per ml) was utilized in this study. 5% Methylene blue (w/v) were prepared for staining of vaginal smears.
Animal
Total of 48 female and 24 male albino mice (12_16 weeks old) were utilized, and the experiments were carried out in the animal house unit at Biotechnology research center / University of Al-Nahrain. Also, the procedure was approved by the ethics committee in the center and under ethical
approval no. (E.B.13). Male and female albino mice were divided separately and equally into six groups including control. All animals were provided with fresh drinking water and fed ad libitum for two weeks before applying protocols.
Experimental design
Synchronization of estrus was performed utilizing prostaglandin F(PGF); and or progesterone (P4), with or without equine chorionic gonadotropin (eCG) in five protocols. Total of eight female mice per group were utilized. Adult albino male mice were introduced into groups in a male/female ratio 1:2 and for four days after day0. Vaginal smears were carried out to confirm estrus phases before applying synchronization protocols at day-3 and before introducing males at day1. Female mice were monitored twice daily for appearance of vaginal plugs after introducing males. Four days later, female mice transferred into new cages and bedding, and monitored for pregnancy and delivery. The dams were separated and gender of litters examined two weeks after delivery.
Synchronization protocols
In the first day (Day-3), Prostaglandin (D-Cloprostenol) (with or without eCG) or Progesterone was administered intraperitoneally (IP). Except GI group (given Normal saline (NS)), female mice were injected prostaglandin with or without eCG at Day0 (Fig. 1).
Vaginal Smears
To determine estrus phase in female mice, vaginal cytology was carried out twice. First, at the day before applying ES protocols, and second, before introducing males. Samples were collected from female mice by modified method using inoculation loop instead of swap technique (Fig. 2).
Briefly, vaginal smear was prepared by mixing the sample with 15 pl drop of normal saline (0.9 % NaCl) on a glass slide. The drops were then thinly spread on the slides and allowed to dry at room temperature (RT). After that, 5% Methylene blue were spread on slides for 20 min for staining, then rinsed gently with distilled water and allowed to dry under RT. The smears were studied using light microscope under 40 x magnifications (Fig. 3).
Days -3 I 0 I 1 I
Groups PGF2a eCG P4 PGF2a eCG i
GI _ - - _ _
GII + - - + + E
GIII + + - - -
GIV + - - + _ '0 s
■s
GV - - + + + e
GVI - - + + - e HH
Fig. 1. Experimental study groups and synchronization protocols
Fig. 2. Steps of modified vaginal smear using inoculation loop. (a) Burning the top part of inoculation loop and cold it with distal water (b), (c) sample collection, (d) mixing with drop of normal saline on slide, (e) spreading the sample on the slide and allow to dry at RT, (f) apply methylene blue and (g) allow for staining for 20 mins, (h) gently washing slides with D.W
a
b
c
d
Fig. 3. Microscopic appearance of vaginal smears of estrus phases in mice. (a) Proestrus, small round nucleated epithelial cells; (b) estrus, presence of anucleated keratinized epithelial cells; (c) metestrus, presence of neutrophils with anucleated keratinized epithelial cell, (d) diestrus, presence of large number of neutrophils
Statistical Analysis
The data of the current study were analyzed using Minitab software version 18. ANOVA test using fishers approach for LSD and two proportion methods were utilized to evaluate the significance of the differences between groups.
Results
Phases of estrus
The results of the current study show that higher proportions of mice before synchrony are in diestrus phase. Also, administration of prostaglandin reduces the proportion of mice being in diestrus before mating (Fig. 4).
Proestrus
Estrus
Й о
tí о a
£
□ Befor Synchony
7S ВО 4S ЗО 1S О
I Before Mating
0 a о
s_
Q_
□ Before Synchrony
7S ВО 4S ЗО 1S О
I Before Mating
GI GII G I I I GIV GV GVI
GI G I I GI I I GI V GV GVI
Й о
'-S
о a о
£
Metestrus
□ Before Synchrony
75 60 45 30 15 0
I Before Mating
Й о
'-S
о a о
£
Diestrus
□ Before Synchrony
75 60 45 30 15 0
I Before Mating *
GI GII GIII GIV GV GVI
G I GI I GI I I GI V GV GVI
Fig. 4. Estrus phases before synchronization (light bars), and before mating (dark bars). Asterisks represent significant statistical difference at p < 0.001
Mating and delivery rate The proportions of mice that show vaginal plugs are significantly different in GII and GIV comparative to control group (p < 0.001). Also, the proportion of mating is different in GVI (p < 0.001). Except GIV, there is no significant difference between groups in the proportion of delivered dams (Fig. 5, right).
Number and gender of litters The results show no significant differences in the average number of litters per dam comparative with control (Fig. 6, left). However, there is a difference between GIII and GV (p = 0.048). On the other hand, data reveal no significant difference in the proportion of male and female litters between groups (Fig. 6, right).
D1 D2 D3 D4
80
60
s o
o a
o ^
Ph
40
20
100
a o
'-B
o a
o —
Ph
□ Mated ■ Delivered p<0.001
75
50
25
T
I
n
I
m
GI G I I G I I I GIV GV GVI
Fig. 5. Proportion of mice showing vaginal plugs after introducing males from Day1 to Day 4 (left). On the right, proportion of mated (light bars) and delivered dams per group (dark bars). Statistical difference is significant at p < 0.001
Average
16
12
J
ab T 111
GI GII GIII GIV GV GVI
A
o
'-S
o a o
s^ Cm
40
20
□ male ■ female
80
60
GI GII GIII GIV GV GVI
Fig. 6. Average number of litters per dam (left), proportion of male and female litters (right). Different letters represent statistical difference at p < 0.05
Discussion
In this study, estrus synchronization is not fully achieved after hormonal application between day-3 and day1. This might be because of the short period of time of estrus that goes unnoticed. Also, frequent vaginal smears might affect the cytology results through stimulation of inflammatory cells (Gal et al., 2014). Therefore, in this study estrus was evaluated by study of disparities of estrus phases between groups before synchrony at day-3 and before mating at day1. There were trending to regular cycling between groups after hormonal application comparative to the status of irregularities before synchrony. It was reported previously that persistent diestrus might be a consequence for housing of mice in large population (Van Der Lee & Boot, 1955). Therefore, administration
of prostaglandins reduces number of mice being in diestrus. This means that application of prostaglandins is of value in lysis of corpus lu-teum and resuming of estrous cycle in mice.
In the current study, mating was evaluated through the appearance of vaginal plugs after introducing male mice. Around 70% of females that were received prostaglandin, show plugs within four days after introducing males. However, around 68% of females, that were received progesterone, did not show plugs within the same period of time. In previous study, the use of double dose of progesterone leads to pseudo-pregnancy in mice (Skliarov et al., 2021). Also, removal of the negative feedback mechanism of progesterone is followed by gonadotropin surge in animal. However, in this study, majority of females that were received
0
0
a
b
8
4
0
0
I/P injection of progesterone did not show vaginal plugs. It might be possible that the current dose of progesterone might be insufficient to induce appropriate NFB, or disrupt gonadotropin surge. Another explanation might be the presence of low level of estrogen in these animals which affect the positive feedback of estrogen on gonadotropin surge (FSH and LH) (Al-Mu-ala & Al-Jiboori, 2010).
In term of pregnancy and delivery rate, the attraction preference behavior through major urinary proteins (MUPs) and copulation depend on pheromones that are synchronized with ovulation to ensure the success of pregnancy (Dey et al., 2015; Hellier et al.., 2018). Despite few studies reported that no effect of the cage size on reproductive performance (Whitaker et al., 2007). In the current study mice show estrous cycle irregularities. This might be due to the stress of being housed in cages (Merkwitz et al., 2016). Also, it was reported previously that keeping pregnant female mice in restricted cages leads to higher miscarriage rate (An et al., 2021). In their study, (Borak & Kohl, 2020) refers to a similar to Bruce effect in rodents and possible link of olfaction and miscarriage in human that exposed to the scent of non-spouse male. In the current study, the proportion of dams that being pregnant was very low. It was previously detailed that the scent of alien male
and not stud male urine in bedding passes through vomeronasal organ (VNO) and main olfactory bulb (MOB) during 3 dpc (day post-coitus). This prevents implantation and pregnancy in mice mediated by diminishing of pro-lactin that led to luteolysis of corpus luteum (Serguera et al., 2008).
Conclusions
Administration of prostaglandin with eCG induces estrus and increases mating rate in female mice. Also, low delivery rate had attributed to the presence of more males within the same cage, and change of bedding once, at the day of males removal, rescue pregnancy of later mated females. Further study to evaluate the success of pregnancy and considering the size of cages and frequency of bedding replacement is proposed.
Declaration of interest
The authors declare that there is no conflict of interest.
Acknowledgments
This work is self-founded. However, all experiments were carried out in Biotechnology research center (BRC). Also, thanks to workers in the animal house unit whom manage animals along the period of this study.
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