Conducting successful artificial insemination (AI) by laparotomy in African lion (Panthera leo)

doi:10.21203/rs.3.rs-3886433/v1

Assisted reproduction technologies including artificial insemination could play an important role in preserving the population of the vulnerable species. In this study, we collected semen from two male lions by using urethral catheterization under general anesthesia. The provided semen was evaluated and a qualified sample with a volume of 0.4 milliliters was extended in a 1:4 ratio by using 1.6 milliliters of Tris-egg yolk-based extender. Stimulation of ovaries in three lionesses of the study was done by administration of 1000 IU of eCG. One hundred hours later, the expected signs of estrous were evident and 750 IU of human Chorionic Gonadotropin (hCG) was administered to induce ovulation. AI was conducted 36 hours after hCG injection through mid-line laparotomy and injecting 0.25 cc of diluted semen with a concentration of 1×10⁶ spermatozoa into each uterine horn by using an angiocath. Exanimation of the uterine horns of the inseminated lionesses was done 60 days after insemination by ultrasonography. It was found that one lioness out of three (33%) was pregnant and had a twin in her uterine horns. The pregnancy lasted for 115 days and two cubs were born by the pregnant lioness.

African lion

Artificial insemination

Laparotomy

eCG

Semen

The reproductive physiology of female African lions (Panthera leo) is similar to other felidae species and their estrous cycle has four phases, consists of proestrus, estrus, diestrus (pregnant or non-pregnant luteal phase) and interestrus. The estrous lasts 2–9 days and its indicating signs are restlessness with increased vocalization and aggression, rolling on the ground and rubbing to the objects together with increased receptivity and tendency to male lions by watching, tracking and contacting them. The age of puberty in this species is about three to four years and the ovarian cycle occurs every 2–3 weeks or every 17 days on average [3]. Similar to leopards, their ovarian activity dose not influenced by season, but the annual pattern of reproduction is influenced by several ecological and environmental factors such as prey biomass [13]. Although they are generally considered as an induced ovulatory mammals, there are several reports about the possible occurrence of spontaneous ovulation and a complex of physical and psychological stimuli play role in this regard [15]. Based on previous reports, the reproductive efficiency is not high, as it's estimated that the litter size per year is around 0.5 and more than 3,000 copulation is needed for every cub that survives over one year. Moreover, the interbirth interval is quite long, lasting about twenty to thirty months. Considering this, lionesses tend to have cubs every two years with an average litter size of three. Once pregnancy establishes, it lasts about 110

days to the time of parturition. In cases of occurring ovulation and failure of conception, pseudopregnancy may develop and lasts about 35 days to nearly two months [14].

As a result of manmade ecological changes and illegal hunting, the population of African lions has steadily decreased over the recent decades and nowadays they are considered as a vulnerable species. Considering this, a higher rate of fecundity is required in order to rapid compensation of mentioned population loss. Fortunately, compare to other species of wild felids, African lions show high breeding success in captivity, an issue that facilitate application of assisted reproductive technologies (ARTs) for preserving their population [7, 10, 22]. In addition, one of the most challenging consequences of a declining population is the higher risk of inbreeding in small groups of inhabitants preserved in geographically isolated areas, including separate pride living in protected zones or individuals remained in captivity [2, 18]. Detrimental effects of inbreeding on the wild felids population is evident, especially in extreme cases, what has been described for cheetahs (Acinonyx jubatus). Inbreeding increases the risk of extinction due to reproductive failure related to reducing semen quality, decreasing fecundity and declining survivability of newborn cubs [21]. A direct correlation between genetic diversity and quality of semen was showed and the rate of poor quality semen in pulations with intense inbreeding is significantly higher [6]. Again, assisted reproductive technologies including Artificial insemination (AI) and semen cryopreservation could provide an opportunity to avoid this condition by facilitation of breeding with novel male lions and entering new alleles in the genetic pool [12, 20].

In this study, we used laparotomy with minimally invasive surgery for insemination of African lions with fresh semen after induction of estrous and ovulation. This study was done in purpose of domestication of this modified technique of AI in order to preserve the population of endangered domestic wild felids like Iranian cheetah and leopard in Iran.

Animals

Two, 9 and 19 years old male lions and three, 8, 8 and 7 years old multiparous lionesses were participated in this study. During the time of the study, the male and female lions were kept in the separate cages, providing standard living condition in captivity for them. Based on nutritional guidelines, they were fed with hog meat, chicken meat and slaughtered cattle carcass up to 5–6% of their body weight in order to provide energy over 130 kcal per kilograms of their body weight as the maintenance energy requirement. They were apparently healthy without any clinical signs of illness and they had moderate body condition score. Two months before conducting the study, administration of antiparasitic treatments regimen was done by single extra label subcutaneous injection of ivermectin (Iver 1® Rooyan darou) at a dosage of 0.3 mg/Kg and adding up deworming compound contained niclozamide (200 milligrams per grams) and pyrantel pamoate (100 milligrams per grams) at a dosage of 5 grams per 20 kilograms of body weight to the feed once a week for three weeks. In order to evaluate the general health of female lions which might affect their fertility, blood sampling and a complete blood count (CBC) analysis was done just before the time of their insemination.

Synchronization of estrous

Diagnostic ultrasonography (US) of the reproductive system of the three lionesses was done by applying 7.5 MHZ ultrasound (KX5000V, Keebomed, Inc) for ruling out pregnancy and possible causes of infertility including ovarian cysts and uterine complications. Considering previous reports about controlling ovarian function in felids [4, 19], intramuscular injection of 1000 IU (International Units) of eCG (GONASER®, HIPRA, Spain) into gluteal muscles wasdone by using of a remote dart gun. Around 100 hours later, they appeared signs of estrous including restlessness, vocalization and rubbing to the cage. In order to sexual stimulation, potentiating signs of estrous and definitive verification of estrous, a mature male lion was transferred to the juxtaposition cage of female lions. After verifying commencement of estrous by observing behavioral signs of both male and female lions such as rubbing

to each other behind the bars of the cages, male restlessness and roaring and tracking females to their cage entrance, 750 IU of human Chorionic Gonadotropin (hCG, IVF-C ®, LG life sciences CO, South Korea) was injected intramuscularly by the same procedure for induction of ovulation. Artificial insemination was done 36 hours after injection of hCG.

Semen collection, dilution and analysis

Semen collection was done from both males after induction of general anesthesia (Fig. 1) by an intramuscular injecting combination of 2 mg/kg ketamine 10% (Alfasan, Netherland) and 0.07 mg/kg medetomidine (Syva, Spain). Around 15 minutes after induction of general anesthesia, the penis was extruded and cleaned and urethral catheterization was accomplished by inserting of sterile commercial dog urinary catheter into the urethra, based on the previously described method [11, 17]. Purposing better capacitation of spermatozoa, provided semen was diluted by Tris- 20% egg yolk solution in a 1:2 or 1:3 ratio depends on the concentration of spermatozoa and after conducting microscopic analysis, remaining diluent solution containing Ham's F-10 culture media and BSA was added to the mixture of semen and Tris to reaching a final concentration of semen to extender ratio of 1:4. Microscopic analysis of semen for morphologic evaluation of spermatozoa and assessment of their progressive motility was done by placing 10 microliters of diluted semen on a pre warmed glass slide under ×400 magnification of a standard light microscope (Nikon, Japan).

Artificial insemination

In order to confirm the accurate time of insemination, plasma progesterone (P4) level and vaginal smear were analyzed. For this purpose, general anesthesia was induced in three female lions of the study by the same producers of male animals (2 mg/kg ketamine 10% and 0.07 mg/kg medetomidine). After induction of general anesthesia (Fig. 2), blood sampling and vaginal smears specimens were taken, according to the following procedures. Blood sampling was done by using heparin contained vacationer tube from the ramus caudalis (saphena leteralis) vein. For taking a vaginal swab, vulvar labia was separated by a gloved hand and a cotton tipped swab was inserted dorsally into the vagina to avoid entering the external urethral orifice. The cotton swab was rotated against the vaginal wall and then rolled two times on the clean glass microscope slide. Prepared slides were air dried at room temperature (25 ^oC) and then fixed and stained according to the modified Wright-Giemsa method [4, 5].

After the end of sampling, lionesses were transferred to the previously prepared site and were prepared for insemination by shaving their abdominal area, complete surgical scrub of skin site of the surgery by using povidone iodine and 70% isopropyl alcohol and placement of sterile surgical drapes. A minimally incisive laparotomy incision of the abdominal layers with a length of 5 centimeters was created in the midline of the abdomen over the linea Alba. After reaching the abdominal cavity, the uterine horn and ovaries were positioned and for confirming ovulation, ovaries were examined by gross observation. Regardless of ovarian status, AI of all three lionesses was done at the tip of both uterine horns, near to uterotubal junction by using a gauge 24 (24 G) angiocatheter and injecting the total number of 2×10⁶ spermatozoa equally into two uterine horns. Following AI, the incision was sutured by ployglycolic acid (PGA) and silk suture material with a metric gauge of five. Prophylactic antibiotic treatment was administered by intramuscular injection of long act gentamycin and amoxicillin compound. Both male and female were recovered from anesthesia by intramuscular injection of an α₂ adrenergic receptor antagonist named Atipamezole (Alzane®, Syva, Spania) at a dosage of 5 mg per each milligram of previously administered medetomidine at the time of induction of general anesthesia.

Pregnancy diagnosis

After induction of general anesthesia by 2 mg/kg ketamine 10% and 0.07 mg/kg medetomidine, ultrasonography was done around 60 days after insemination, for diagnosis of pregnancy.

Microscopic analysis of semen collected from 19 years old lion, showed that less than two percent of spermatozoa had progressive motility and nearly all of them had major defects like coiled tails with side to side shaking movements. Considering this, it was discarded as it was disqualified for being used in insemination. Around 0.5 cc of thick semen was recovered from a younger, 9 years old lion. Sperm concentration after initial dilution showed that it has25 × 10⁶ spermatozoa per milliliter, 75% of them were alive based on the eosin-nigrosine staining method and 55% had progressive motility. Around 35% of spermatozoa had circling movement with various defects including the coiled tail, headless and droplets. This range of defected spermatozoa is normal in felids [1]. The volume of 0.4 ml of semen was added to 1.6 milliliters of the extender to provide 1:4 dilution and 0.25 milliliter of diluted semen (~1×10⁶ spermatozoa) was injected into each uterine horns of the inseminated lioness.

CBC, DIFF and hematologic analysis of lioness in this study were normal (Table 1). The concentration of P4 in three female lions, just before insemination is presented in Table 2. According to provided results, the appropriate time of insemination was confirmed by increased P4 level to over two nanograms per milliliter (ng/ml) and microscopic observation of keratinized superficial epithelial cells with a pyknotic nucleus (Fig. 3). Gross observation of ovaries during laparotomy showed that lioness number one had one corpus haemorrhagicum (CH) and two follicles with diameters of 0.3 and 0.4 centimeters in the right ovary and two CH in left ovary. Lioness number two had one follicular cyst with diameter of 1.5 × 2.7 centimeters in right ovary while she's left ovary was static, without follicles and CH. Lioness number three had one CH and one follicle in right ovary and two CH in the left ovary (Table 2).

Two months after insemination, lioness number three showed signs of mammary glands hypertrophy and reduction of appetite and movement. Ultrasonography examination of the uterus revealed twin pregnancy with the presence of two fetuses in the uterus. Lioness number one was non-pregnant and lioness number two had signs of pseudopregnancy and mammary gland hypertrophy with cystic corpus luteum in right ovary. Parturition in a pregnant lioness occurred 115 days after the time of insemination and twin cubs with different sexes were born by her. Unfortunately, male cub was lost due to mismothering, but female cub survived (Fig. 4).

The results of analyzing CBC and DIFF of inseminated lionesses showed that all hematological and blood indices were within the normal range and verified that the animals had an appropriate health condition. The procedure of hormone therapy for ovarian stimulation and induction of ovulation in our study was done by using the explained eCG/hCG treatment protocol. The hormonal treatment regimen and dosage in this study was inspired from previous reports about eCG/hCG application in lion and other wild felids [8]. Alternative protocol of hormone therapy by using porcine FSH/LH [7] and also analogue of gonadorelin releasing hormone (GnRH) for induction of ovulation was recently reported for this purpose [4], but due to approved efficacy of traditional eCG/hCG protocol, we preferred to use this protocol. The number of surged follicles (n = 3) after administration of eCG was lower compare to previous reports and this might occurred because of low ovarian activity in the lionesses took part in our study.

Sperm collection was done by urethral catheterization, this method was used because of its reported advantages including higher quality of collected semen and reduced risk of urine contamination compare to other methods such as electroejaculation [10]. Poor quality of collected semen from the aged male lion might be related to congenital or acquired causes of infertility or resulted from the age dependent decline of sperm quality, which is reported in other species, too [9]. Considering the normal size and of texture of testes during preliminary breeding soundness examination, the second hypothesis is more possible. The volume of collected semen (0.5 mL) from the younger lion was within the previously reported range (0.1–0.7 mL) of collected semen volume through urethral catheterization. The percentage of progressive motile spermatozoa was normal but the concentration of spermatozoa

was lower (25×10⁶) than previous reported range in samples collected by urethral catheterization (125×10⁶ – 4860×10⁶) and was whiten the range of those reports collected semen samples by electroejaculation (4.5×10⁶ – 956×10⁶) [4, 11].

Surgical insemination of lion by conducting laparoscopy and non-surgical AI method by using urinary catheter was reported before, but as far as we know this is the first report of AI in this species trough mini-invasive laparotomy. Each mentioned method of AI has its own pros and cons. Laparoscopy approach needs expensive equipment. Moreover, technical proficiency of working with laparoscopy instruments is essential for achieving optimum result. Otherwise, in the non-surgical method, passing the catheter into the cervical ostium is difficult as it is not possible to manipulate cervix and fix its position manually by transrectal or transabdominal palpation due to cranial position of the cervix and large abdominal cavity of this species, respectively. This difficulty reduce the risk of successful insemination and hence lower conception rate by this method is predictable [4]. Successful application of minimally invasive technic of laparotomy to intrauterine insemination was reported in other animals like ewes [16]. Easier intrauterine injection of spermatozoa compare to laparoscopy because of diminished mobility of uterus and hence higher chance of pregnancy due to lower risk of errors in intrauterine injection of spermatozoa, uterine damage and adhesion was reported as the advantages of laparotomy for conducting AI.

Reported pregnancy rates in non-surgical method of AI by using urinary catheter and laparoscopy was around 28%.Considering low number of inseminated lions in conducted studies, comparing pregnancy rates between reports has limited values. However, it should be noted that the pregnancy rate of 33% in our study was achieved with a 25 fold lower minimum concentration of spermatozoa and inseminated lionesses were inseminated after induction of estrus rather than natural estrus [4]. Results of this study suggest that our method of AI through laparotomy can be successfully applied, especially in cases of lacking of enough concentration of spermatozoa which is needed for other method of AL. The number of born cubs and 115 days duration of pregnancy was similar to the range of previous reports (105–114 days).

Ethical approval

Animal welfare condition and all medical interventions in this study was conducted according to code of ethics of the faculty of veterinary medicine of University of Tehran (IR.UT.VETMED.REC) and approved by biomedical research ethics committee of this faculty.

Availability of data and materials

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Conflict of interest

There is no conflict of interest.

Funding Statement

The conducted research has received no funding.

Acknowledgment

Special thanks to the Meral veterinary clinics' staff for providing equipment and excellent technical support and the manager of the sari zoo Dr. Ali Bagheri for his collaboration on this project.

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Table 1. Average of CBC and DIFF values of three lionesses
Indices	Results	Normal ranges	Units
WBC	20.3	7.2-26	× 10³/ ul
Segmented neutrophils	61	60-85	%
Lymphocytes	32	7-33	%
Monocytes	1	1-9	%
Eosinophils	6	1-6	%
Basophils	0	0-2	%
RBC	6.82	5.1-8.4	× 10³/ ul
Hemoglobin	10.6	8.9-14.6	g/dl
Hematocrit	33.2	26.8-44.1	%
MCV	48.7	46.6-60	fl
MCH	15.5	14.8-19.1	pg
MCHC	31.9	29.6-35.7	g/dl
Platelets	172	155-627	× 10³/ ul

Table 2. Concentration of serum P4 level at the time of insemination.
Animals	P4 level (ng/ml)	Right ovary	Left ovary	Interpretation
Lioness no. 1	3	1 CH, 2 Follicle	2 CH	Concentration of P4 during follicular stage is less than 1 ng/ml, and it increases at the time of ovulation (2-25 ng/ml)
Lioness no. 2	1.4	Follicular cyst	no
Lioness no. 3	17.2	1 CH, 1 Follicle	2 CH

No competing interests reported.

Conducting successful artificial insemination (AI) by laparotomy in African lion (Panthera leo)

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Synchronization of estrous

Semen collection, dilution and analysis

Artificial insemination

Pregnancy diagnosis

Results

Discussion

Declarations

References

Tables

Additional Declarations

Status:

Version 1