Comparative Study of Different Assisted Hatching Techniques and In Vitro Culture Media on Mice Embryo Hatching Rate

Research Article

J Bacteriol Mycol. 2021; 8(1): 1163.

Comparative Study of Different Assisted Hatching Techniques and In Vitro Culture Media on Mice Embryo Hatching Rate

Negota NC1, Mphaphathi ML3, Nethenzheni LP1, Rammutla TL1, Serota NR1, Barry DM1 and Nedambale TL2,3*

1Department of Animal science, University of Venda, South Africa

2Department of Animal Science, Tshwane University of Technology, South Africa

3Germplasm Conservation and Reproductive Biotechnology, Agricultural Research Council, South Africa

*Corresponding author: Nedambale TL, Department of Animal Science, Tshwane University of Technology, Germplasm Conservation & Reproductive Biotechnology, Private Bag X680, Pretoria, 0001, South Africa

Received: January 25, 2021; Accepted: February 17, 2021 Published: February 24, 2021


The study investigated the influence of Assisted Hatching (AH) techniques (mechanical, chemical, enzymatic thinning and laser) and two in vitro culture media (Ham’s F10 and TCM-199) on hatching rate of mice embryos (blastocyststage) following 24 hours of culture. The C57BL/6-black (n=30) and BALB/cwhite (n=30) mouse breeds were raised until maturity and naturally bred to produce a F1 generation. The light in the breeding house was controlled and the mice were fed ad libitum. Female mice (n=30) were injected (peritoneal) with 0.1 ml (5 IU) of eCG into the abdominal cavity with 1 ml syringe and 0.5x16 mm needle to stimulate follicular growth and 46-49 h later was injected with 0.1 ml (5 IU) of hCG to cause ovulation, maintain the corpus luteum and stimulate it to secrete progesterone for maintenance of pregnancy. After the injection, the male and female (1:1) could mate overnight. Female mice with vaginal plugs were observed and kept separately for blastocyst-stage embryos collection on day three following successful mating. They were euthanized, and abdomen sterilized with 70% ethanol. Using a sterile surgical scissor, a fine cut was made, holding the skin firmly above and below the incision, the skin was pulled apart using forceps. The embryos were flushed from the uterus using a 30-gauge needle with culture media. Following the AH techniques, embryos were cultured in TCM-199 or Ham’s F10 for 24 hours and zonal thickness of all hatched embryos were measured. Immediately after assisted hatching, the embryos were cultured into two different in vitro culture media. All embryos hatched were stained and the zonal thickness of embryos were measured. The number of blastomeres were counted and recorded 24 h later. Data collected were subjected to analysis of variance using PROC General Linear Model. The Tukey’s test was used to separate the means. A significant difference was observed between the thickness of Zona Pellucida (ZP) pre and post treatment after 24 hours of culture. However, there was no significance difference among blastocyst hatching rate and the blastomeres nuclei counted after staining. The thickness of the ZP decreased with individual AH techniques. The interaction between AH techniques and in vitro culture was found to be significantly different on blastocyst hatchability. However, laser AH technique had highest hatchability (56.3%) when embryos were cultured in TCM-199 followed by mechanical AH techniques (52.6%). The hatchability rate (33.3%) was recorded in the chemical AH technique group. The blastomeres nuclei counted under interaction of AH techniques and culture media was not differently affected, with the values ranging from 69 to 76%. In conclusion, the use of different AH techniques resulted in varying effect and increase outcomes towards the hatching rate.

Keywords: Blastocyst; Assisted Hatching; Mouse; Zona Pellucida; Ham’s F10; TCM-199


Biologically, after an embryo has reached the blastocyst-stage, the Zona Pellucida (ZP), becomes thinner and groove appears allowing blastocyst to hatch and implants to the uterine wall. The hatching of the blastocyst is the crucial step that must take place before implantation. The failure of ZP rapture impairs the hatching of the blastocyst, thus limiting conception rate [1]. Assisted Hatching (AH) is one of the assisted reproductive technology methods that artificially manipulates the ZP. The use of AH methods has been reported to be essential for improving implantation rates of embryos in both animals and humans [2].The use of different AH techniques to create a hole in the ZP or weaken the ZP at the blastocyst-stage of embryo development has been previously documented [3]. Furthermore, it allows easy hatching of the blastocyst from the ZP [3,4] recorded an improvement in pregnancy rate after the use of laser AH technique in embryos. Assisted hatching can be done by a variety of techniques, including the use of chemical, mechanical, enzymatic and laser assisted hatching techniques [5,6] reported that AH techniques addresses the challenge of embryo hatching from the ZP by improving the potential of blastocyst-stage embryos to hatch in vitro.

Mechanical AH technique is done by dissection and creating an opening in the ZP of the blastocyst, while the embryo is held stable with a holding pipette. The ZP is then pierced with a micro needle through the space between the ZP and blastomeres. Acidified tyrode’s solution is used for the chemical AH technique and it has been previously reported in detail by [6]. The enzymatic mechanism is enzymatic digestion of the ZP using trypsin enzyme. The laser AH technique uses a laser beam to drill the outer part of the ZP and can be done with high precision and repeatability. It is reported that there is no side effect on embryo development. These techniques have been used, but there is information on which of the method is the most effective when used with which type of in vitro culture media. It was reported that AH can exert both a beneficial as well as a deleterious effect, on subsequent embryonic development depending on several factors. These factors include, ZP thickness, the size of the hole created, mechanical damage to the embryo by manipulation, chemical damage by acid solution and the technical skill of the operator [7].

The ZP is layer composed of glycoprotein, carbohydrates and zona pellucida-specific proteins that cover the oocytes before fertilization until hatching of the embryo. Before hatching, the blastocyst should undergo a series of contractions and expansions that cause a decrease in the thickness of the ZP until it becomes almost invisible [8]. But this process is hindered by the increase in the ZP thickness of the embryo which in turn affect the process of embryo hatching [9]. The hardening or thickening of the ZP after fertilization was proposed as the one that limit hatching rate in mammalian embryos [10].

Failure to hatch has been largely attributed to ZP hardening and suboptimal culture conditions. In vitro produced embryos may result in hardening of the ZP that is resistant to thinning and subsequently impair the hatching process, whereas, in vivo produced embryos may result in loss of uniformity and thinning of ZP, which precede to hatching. Sub-sequential culture media are also used to increase blastulation and pregnancy rates in assisted reproduction treatments.

In vitro processes involve the use of culture media, which is essential in the maturation and development of embryos. The constituents of the culture medium have got an effect on the cultured embryo development. Biotechnology laboratories are using lot of different in vitro culture media and the selection of good, reliable and clean media is a necessity for their activities [11]. Reported on the use of TCM-199 that showed an increase in blastocyst formation of embryos during in vitro culture. There are differences in the degree to which these media support embryos development. However, the reviewed information concerning (TCM-199 and Ham’s F10) is that they are all good enhancers of high fertilization [12]. Authors hypothesized that AH can enhance hatching rate of the blastocyststage embryos, by both catalyzing the time and rate of hatching. Therefore, the present study was conducted to compare four AH techniques (mechanical, chemical, enzymatic thinning and laser) and use of two in vitro culture media (Ham’s F10 and TCM-199) on hatching rate of mice embryos (blastocyst-stage) following 24 hours in vitro of embryos in different groups per droplet.

Materials and Methods

Study site

The study was conducted at the Centre of Excellence in Assisted Animal Reproduction, Biotechnology Laboratory, School of Agriculture, University of Venda, Thohoyandou, Limpopo, South Africa. Coordinates are as follow (22° 58’ S, 30° 26’ E). Daily temperatures at Thohoyandou vary from about 25°C to 40°C in summer and between approximately 12°C and 26°C in winter. Rainfall is highly seasonal with 95% occurring between October and March. The average rainfall is about 800 mm but varies [13].


The culture media used in this study were Ham’s F10 and TCM- 199 (Sigma). The hormones were eCG (Sigma), hCG (Sigma), Ham’s F10, TCM-199 and Tyrodes solution (Sigma).

Experimental design

Three factors were investigated. These were AH techniques and in vitro culture medium culture. The first factor involved four AH techniques; Number of AH (control), laser, mechanical, chemical, and enzymatic. The second factor involve in vitro culture media; TCM-199 and Ham’s F10. The third factor involve embryo grouping per 50μl droplet; one, two, three and four embryos per drop. The experiment was set up as 4x2x4 factorial design arranged in a Completely Randomized Design (CRD) to determine the significant differences.

Management of mouse

Mice were housed in cages with sawdust. They were raised and fed with mouse pellets and water ad libitum. The sawdust in the cages was changed every 5 days. The light in the breeder house was regulated day and night. The light was on from 5 am until 7 pm (14 hour) and was switched off from 7 pm until 5 am (10 hour). A constant temperature of 24°C was maintained in the breeder house.

Production of F1 generation

Black female mice (C57BL/6) were mated with white male mice (BALB/c) and were kept together in the breeding cage for 7 days. After 7 days the males were removed and returned to their original cages. The F1 offspring were born 21 days after mating. The offspring were raised until they were 6 weeks old.

Preparation and Administering of Injections for F1


Folligon is available in a bottle of 1000 International Units (IU) each (Folligon, Intervet SA, MSD Animal Health). One bottle of 1000 IU was diluted with saline using a syringe to give a working concentration of 50 IU/ml = 5 IU/ 0.1 ml. The solution was stored as 5 IU in 1.0 ml syringes and kept frozen at -30°C.


Chorulon is available in a concentration of 1500 (IU) of hCG (Chorulon, Intervet SA, MSD Animal Health). This was dissolved in the saline solvent. The Folligon was brought to a concentration of 50 IU/ml=5 IU/0.1 ml. The solution was stored as 5 IU in 1.0 ml syringes and kept frozen at -30°C.

Female mice (n=30) were injected (peritoneal) with 0.1 ml (5IU) of eCG into the abdominal cavity with 1 ml syringe and 0.5x16 mm needle, and 46-49 h later, 0.1 ml (5 IU) of hCG was injected. The eCG was used to stimulate follicular growth and hCG was used to mimic Luteinizing Hormone (LH) to cause ovulation, maintains the corpus luteum and stimulates it to secrete progesterone for maintenance of pregnancy. After the injections, the F1 male and female mice (ratio of 1:1) could mate overnight and the next morning the vaginal plugs were observed to confirm mating. The mice with vaginal plugs were kept separately for the collection of blastocyst-stage embryos on day three following successful mating.

Blastocyst stage embryo collection

On day three after successful mating, the mouse was taken from the cage and placed on the cage bars, so that it can grip the bars with its front paws. The cervical vertebra was separated by applying firm pressure at the base of the head (back of the neck). The mouse was laid on its back on the absorbent paper and its abdomen soaked with 70% ethanol to maintain sterility. A fine cut was made in the middle of the abdomen by using a sterile surgical scissor, while holding the skin firmly above and below the incision. The skin was pulled apart using watchmaker forceps. The blastocyst-stage embryos were collected from the uterine horn by flushing the embryos using the D-PBS loaded in the tip of a pipette inserted onto a 1 ml syringe. A 26-gauge needle was inserted into the tip of the uterine horn (from the ovarian side) to flush blastocyst embryo towards the uterine body. A 1 ml syringe with a pipette tip was used to pick up the blastocyst-stage embryos and washed three times in droplets of D-PBS and culture media as prepared in the tables below.

Mechanical assisted hatching technique (partial zona dissection)

In brief, the 400 embryos were held by a holding pipette held 30° from the left and the Partial Zona Dissection (PZD) injection needle was inserted from a right of the embryo. The ZP of the embryo was pierced with the PZD injection needle with extensive care in order to avoid damage to the inner cell mass. The ZP was pierced with the needle so that a hole is created on it.

Chemical assisted hatching technique (acidified tyrode’s solution)

A concentration of tyrode’s solution with a pH of 2.5-3 in 99.875% of culture medium was used to digest the ZP. The holding pipette was lowered into a drop containing the blastocyst-stage embryos (n=10) to be digested in order to control the movement of embryos. Embryos could rest stable in the petri dish with either TCM-199 or Ham’s F10 culture medium. The injection needle was removed from the packet and the tip of the needle was party broken using a hand to make a hole on it that will allow easy aspiration of acidified tyrode’s solution inside the injection needle. The injection needle was filled with acidified tyrode’s solution. The same injection needle related to the homemade mouth-control delivery system. A volume of 20μl acidified tyrode’s solution was aspirated and a mouth-controlled homemade delivery system was used to blow acidified tyrode’s solution gradually over a small area of the ZP until it is digested. The embryos were immediately removed and washed two times in the TCM-199 or Ham’s F10 culture medium to remove traces of acids.

Laser assisted hatching technique

The dish containing the mouse blastocyst-stage embryos (n=10) was placed on the stage of the inverted microscope and the embryos were located under focus. The laser lens was rotated to the position of the drops and was focused on the first embryo. The laser target was placed over the ZP in a place that had no blastomeres directly underneath. The laser beam was guided by safety circles which help the operator to choose the area of interest and the target was positioned on the ZP and ensure that the safety circle did not overlap the blastomeres. Beaming of the ZP was done with a single medium pulse on the ZP of the all the embryos, without breaking the layer of blastomeres. The same procedure was repeated for all the embryos in TCM-199 or Ham’s F10 medium.

Enzymatic with trypsin assisted hatching techniques

The TCM-199 or Ham’s F10 medium with the concentrations of 0.5% trypsin, pH of 6.8-7.2 and 0.1% of EDTA was used in the digestion of the ZP. Assisted hatching technique using the above medium was prepared in a 35 mm petri dishes and 50μl droplets of trypsin was placed in a petri dish and covered with mineral oil. Embryos (n=10) were placed using glass pipette in the drops for one hour to allow partial zonal digestion. Digestion of the ZP was done under mineral oil in atmosphere at 37°C on the stage on inverted microscope. The measurement of ZP thickness was done using the microscope connected to computer software that allows to place mouse curser on the points of thickness to give readings.

Embryo culturing

After the AH techniques were completed, embryos were cultured in TCM-199 or Ham’s F10. The embryos were cultured following different grouping methods in a 50μl droplets (one embryo in a drop, two embryos in a drop, three embryos in a drop and four embryos in a drop). This was aimed to determine if there is any effect of group embryo culture. The window period of 24 hours of culture was allowed for all embryos to undergo hatching after AH was performed. At the end of 24 hours of in vitro culture, the zonal thickness of all hatched embryos were measured and recorded.

Embryo staining

After 24 hours of embryo culture, the hatched blastocysts were stained with Hoechst 33258. The hatched blastocysts were removed from the culture droplets and placed on a glass slide. The medium was aspirated using a micro-pipette to remove most of the volume of medium. The staining was done in the 4-well plate where well one and well two were filled with 0.5 ml of phosphate buffered saline and polyvinyl pyrrolidon medium, well three filled with 0.5 ml fixative solution and well four filled with Hoechst 33258 of concentration 0.5μg/mL in saline.

The hatched blastocyst-stage embryos were washed in well one and fixed with fixative solution (paraformaldehyde in D-PBS, 40 mg/ml) in well three for 30-60 minutes. After fixation in well three, embryos were stained in well four for a period of 20-30 minutes and washed again in well two before transferred to the slide. Stained embryos were placed on a new slide and labelled. A cover slip was placed to cover the embryo on the slide with Vaseline placed at each corner of the slip. The cover slip was used to cover the embryos to prevent foreign particles and spread the blastomeres.

Stain solution was added under the cover slip to cover the embryo. The stain is kept in the dark place at 2-8°C (SIGMA ALDRICH Co, Intervet South Africa). Freshly made stain was used once per day. The Cutex nail polish (M.A.C Studio Nail Lacquer) was used to cover all the sides of the cover slip to prevent the hatched embryos to escape from underneath the slide. The labelled slide was turned upside down for counting the blastomeres under fluorescent UV light within 4 hours using an inverted microscope under X400 magnification (Nikon eclipse TI, Narishige co., Ltd. USA).

Statistical analysis

Data collected were subjected to analysis of variance using PROC General Linear Model of SAS version 9.4, where samples were randomly collected from the F1 generation mice. Where significant differences between groups were detected, the Tukey’s test was used to separate the means and the following p-values were used (P<0.05; P>0.05 and P<0.01).

Ethical considerations

The ethical clearance certificate referenced (SARDF/15/ ANS/03/0110) was obtained from the University of Venda Research Ethics Committee.


The thickness of the ZP (7.3μm) before AH and after culture for 24 hours (12.8μm) differed (P<0.01) (Table 1). There was no different (P>0.05) on control group regarding the ZP thickness before and after culture. While other techniques showed difference and other showed no difference (P>0.05). Comparing the AH techniques, it was observed that they differed (P<0.05) in hatchability% (Table 1).