Gaia C. Luvoni, DVM, PhD, DECAR
Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Milan, Italy
Assisted reproductive techniques (ART) are aimed to improve reproductive performances. Manipulation of the gametes outside the bodies of the originating organisms is the core of reproductive biotechnologies. Besides artificial insemination (AI), which is widely applied, particularly in dogs, other techniques that apply both sperm and oocyte manipulation in vitro have been developed in several mammalian species including dogs and cats. In vitro embryo production achieved significant advances particularly in cats, and sperm-oocyte interaction in vitro represents a powerful tool for testing male gametes.
Ejaculated, epididymal and testicular spermatozoa can be used in in vitro procedures. In dogs, ejaculated spermatozoa are easily obtained by digital manipulation or with the use of an artificial vagina. In cats, ejaculation can be obtained with artificial vagina only in trained males, whereas in not trained males electroejaculation under general anesthesia and urethral catheterization after administration of medetomidine (Zambelli et al., 2007) have been proposed.
Epididymal spermatozoa are generally obtained from isolated epididymis caudae when a valuable animal dies accidentally or undergoes orchiectomy for medical purposes. In the case of erectile dysfunction or ejaculation failure due to pathological conditions, in vivo collection could be an option. We recently demonstrated in dogs that percutaneous epididymal sperm aspiration (PESA) is a feasible alternative to the retrieval of spermatozoa from isolated organs (Varesi et al., 2012). PESA, firstly developed in men (for review, see Shah 2011), consists in the needle aspiration of epididymal spermatozoa from cauda epididymis through the scrotal skin. Epididymis is not directly visualized and the site of aspiration is guided by palpation. The results indicated that the population of epididymal spermatozoa retrieved by PESA has similar characteristics to that collected from isolated organs, although a wide variation in sperm concentration is observed among animals.
Testicular spermatozoa are retrieved after dissection of tissue from isolated testes and centrifugation of the cell suspension. Testicular spermatozoa are immotile, as the motility is acquired during transit in the epididymis.
Oocytes can be harvested from ovaries isolated postmortem or at ovariectomy. Different methods for oocyte retrieval have been developed (for review, see Luvoni et al., 2005; 2006). Among these, canine and feline oocytes are recovered by aspiration of the antral follicles, mincing of the ovarian cortex and follicle dissection.
Aspiration is performed by using a syringe and needle and puncturing the antral follicles, allowing collection of the follicular fluid together with the oocytes surrounded by cumulus cells. Mincing of the ovarian cortex and follicle dissection are the most common procedures adopted in carnivores. In the first method, the ovarian surface is cut repeatedly lengthwise and crosswise with a surgical blade and the ovaries are washed in a dish filled with culture medium. For follicle dissection, the ovaries are divided into small pieces and the antral follicles are punctured with a needle to release the oocytes into the culture medium.
These oocytes are generally at the germinal vesicle (GV) stage of the meiosis (immature oocytes) and need to be cultured in vitro in order to achieve the metaphase II stage (mature oocytes) before fertilization (IVF).
In vivo collection by laparoscopy or laparotomy, after a previous hormonal stimulation of the female to induce follicular development, allows the retrieval of preovulatory (mature) oocytes that can be fertilized without a previous culture for maturation. This procedure is not commonly used for recovering oocytes in the bitch, because in this species the preovulatory oocytes are still meiotically immature.
In Vitro Sperm-Oocyte Interaction
In Vitro Embryo Production and Transfer
In vitro embryo production, may give a chance to obtain progeny from animals in which reproductive disorders limit conception rates: when migration of embryos into the uterus is impaired, in the case of ovulation failure or permanent anoestrus, and in animals that do not respond to hormonal stimulation.
Embryos can be obtained through in vitro fertilization or intracytoplasmic sperm injection (ICSI).
The first consists in the co-incubation of female and male gametes in specific media. Before the co-incubation of gametes can proceed, it is important that oocyte maturation and sperm capacitation have been completed. After co-incubation, presumptive zygotes are cultured in vitro for embryo development.
ICSI is the injection of a single spermatozoon through the zona pellucida directly into the ooplasm of a mature oocyte and is the first choice when only few normal spermatozoa are available, as in case of poor quality semen samples (oligozoospermia or teratozoospermia).
In dogs, the efficiency of in vitro embryo production is very low and greatly variable. Canine oocytes mature in vitro at a much lower rate than oocytes of other species and after fertilization embryo development is poor. These results are mainly due to the peculiar reproductive physiology of the bitch that, differently from other species, ovulates immature oocytes. Oocyte maturation takes place in 2–3 days in the oviductal environment, rather than in the follicular environment. Low success of in vitro embryo production depends on the low rates of oocytes maturation that can be obtained in vitro (for review, Luvoni et al. 2006; Chastant-Maillard et al. 2010). The only report of intrauterine transfer of in vitro fertilized oocytes goes back to 2001 (England et al. 2001). No other attempts to transfer embryos derived from in vitro fertilization have been reported in dogs.
In cats, in vitro embryo production is more successful than in dogs. In vitro maturation and fertilization of oocytes results in the development of 40–60% blastocysts after culture (Pope et al. 2006a). Laparotomic or laparoscopic transfer of in vitro derived embryos in queens treated with gonadotrophin for estrus synchronization resulted in the birth of kittens (Pope et al. 2006b; Pope et al. 2009).
Successful embryo production from feline oocytes has also been achieved through ICSI with ejaculated and epididymal spermatozoa. The birth of healthy live kittens following transfer of ICSI-derived embryos to synchronous recipients was first reported fifteen years ago (Pope et al. 1998). It has also been showed that testicular spermatozoa from the domestic cat are capable of fertilizing in vitro matured oocytes via ICSI and supporting embryo development to the blastocyst stage (Comizzoli et al. 2006).
Sperm Function Tests
In vitro interaction between spermatozoa and oocytes provides important information on the ability of the male gametes to fertilize. So-called "sperm function tests" allow the evaluation of functional integrity of spermatozoa and contribute to achieve a better diagnosis in case of infertility due to male factors. These tests might also be very useful to evaluate the fertilizing ability of frozen/thawed spermatozoa with the aim of defining an optimal protocol for cryopreservation.
Sperm function tests provide information that cannot be obtained by routine semen analysis based on the evaluation of parameters as sperm concentration, motility, morphology and viability. The gamete interaction in vitro allows the assessment of capability of sperm cells to accomplish early events occurring during fertilization as the binding to the zona pellucida (ZP), the penetration into the ZP, or the penetration into the ooplasm.
Zona binding assay (ZBA) or Hemizona assay (HZA) consists of the co-incubation of capacitated spermatozoa with oocytes or hemizonae pellucidae either fresh, or frozen, or chilled, or stored in a hypertonic salt solution, in which the biological and physiological properties of the ZP are well preserved.
Hemizona has the advantage of providing a comparison of binding capacities between two sperm samples on a single oocyte decreasing the binding variability due to the oocyte itself. After incubation, the oocytes are washed gently to remove loosely attached spermatozoa and the number of spermatozoa firmly attached is counted.
Zona pellucida or oocyte penetration tests (ZPT, OPT) can be performed with homologous mature oocytes or, given that homologous oocytes may not always be available, with heterologous gametes (hamster oocytes).
It has been demonstrated a significant difference between fertile and infertile dogs in the number of tightly bound spermatozoa to the hemizona (Mayenco-Aguirre, Pérez Cortés 1998), and in the cat in vitro tests have been used to evaluate teratozoospermic semen samples (less than 40% normal spermatozoa) (Howard et al. 1991), a condition often observed in Felids. It has been shown that abnormal cat spermatozoa are capable of binding to and entering the outer zona pellucida, but their ability to penetrate the inner zona and reach the perivitelline space to fertilize the oocyte is compromised.
Biotechnologies might be helpful in dogs and cats with low reproductive performances. In cats, kittens have been born after transfer of embryo produced in vitro, while in dogs further investigations are required to achieve the results obtained in cats.
Sperm function tests improve the semen analysis and are crucial when a procedure of assisted reproduction is needed to increase the chances of reproduction in sub-fertile or infertile subjects. For instance, when defective sperm-ZP interaction might impair in vitro fertilization rates, ICSI should be performed. In human medicine, Medically Assisted Procreation techniques are categorized into three levels based on complexity and invasiveness. The physician should select the appropriate procedure, giving priority to the easiest and less invasive technique. Clinical applications of reproductive biotechnologies in dogs and cats should respect the same guidelines. Thus, a careful clinical examination, a diagnosis of the aetiology of infertility, and conventional treatments should be attempted before planning ART. Although not all the laboratories are equipped to perform these techniques in carnivores, an increased diffusion in the near future ought to be done.
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