Volume 7, Issue 10, October – 2022 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Epididymal Spermatozoa Recovery from Post-

Mortem Cattle Preservative Effects of Skimmed Milk,
Coconut Water and Egg Albumin on Caudal
Epididymis Sperm
Abílio Paulo Changule, Maria da Glória Taela, Gildo Macassa, Gaspar Ngulele, Hiracema Inacio
Center for Genetic Resources and Animal Assisted Reproduction
Agricultural Research Institute of Mozambique, IIAM
Maputo, Mozambique

Abstract:- with the objective of comparing the floating, mincing, and flushing (Kang et al., 2018; Raseona
preservative effects of skimmed milk, coconut water and et al., 2018).
egg albumin for epididymal spermatozoa recovered from
cattle after death a floating technique under refrigerated In Mozambique, most of livestock smallholder farmers
conditions (5oC) was employed. For the recovery of are unable to carry out the collection and conservation of
spermatozoa, thirty-two testis-epididymis complexes semen from their males. However, due to indiscriminate
were collected after slaughter of the bulls, dissected and livestock slaughter for human consumption, particularly from
divided into 3 groups for treatments. Motility, vigor and stolen stock, bulls and bucks of high genetic merit and up are
spermatic pathologies were daily analyzed. In all being slaughtered before their potential has been fully used.
treatments was possible to recover viable spermatozoa As a result, there is great waste of genetic material,
with the values of motility and vigor of 25.44± 7.82% and contributing for farmers and country’s economic loss due to
21.22± 6.62% Vs 12.20± 7.87% and 2.22± 0.58% Vs 1.91± unexpected death of males, particularly bulls. In this sense,
0.49% and 0.92± 0.61% for skimmed milk, egg albumin the aim of this study is to evaluate the effect of skimmed milk,
and coconut water respectively. Skimmed milk and egg- coconut water and egg-albumin on recovered epididymal
albumin showed better results, however, there were no spermatozoa.
significant differences between the three preservatives
(P>0.05). The results of this research show that it is II. MATERIAL AND METHODS
possible to preserve recovered bulls’ spermatozoa viable
after death of the donor for a maximum period of 6 days A. Study location
using skimmed milk and egg white and 3 days for coconut The study was carried out in the facilities of the Animal
water. Genetic Resources and Assisted Reproduction Techniques
Center of the Directorate of Animal Science located in Matola
Keywords:- Recovery Spermatozoa; Floating Method; (CRGTRA – DCA / IIAM) and in the Reproduction
Motility; Viability; Bulls. laboratory of the Faculty of Veterinary Medicine in Maputo,
Mozambique.
I. INTRODUCTION
B. Collection and sampling
The Artificial Vagina (AV) and Electroejaculation (EE) The harvest of Epididymis Testicles Complexes (ETC)
are two conventional methods frequently used for semen was carried out at the Municipal Slaughterhouse in Maputo,
collection’s purposes from bulls (Alvarez et al., 2020). Mozambique. The ETC were transported to the laboratory in
However, there are situations that can prevent the use of these an isothermal box with recyclable ice to keep the temperature
methods, such as unexpected death or erectile dysfunction low and avoid compromising the samples. Transport from the
(Prieto et al., 2014; Kang et al., 2018; Pleshkov et al., 2022). slaughterhouse to the laboratory took approximately half an
In these cases, the loss of genetic material from these animals hour. 32 ETC from bulls of different breeds, reared in
is inevitable. The spermatozoa recovery is an alternative different production systems, aged between 2 -8 years old,
method, which has been used in addition to AV and EE, weighing between 250-350 kg were used.
allowing the conservation of germplasm from dead sires and
its use through Artificial Insemination for livestock genetic C. Spermatozoa recovery method
improvement (Cunha et al., 2019; Dziekońska et al., 2020). Caudal epididymis spermatozoa were recovered by the
Furthermore, this method has also been successfully used in floating technique in a Petri dish with a diameter of
several species, both domestic and wild, when by any reason 90x15mm. After dissection, the tail of the epididymis was
they have to be esterilized or killed and thus represents a very sliced and let to rest for 15 minutes in a preservative medium
useful resource for the creation of germplasm banks for futher composed by 20 ml of each skimmed milk, coconut water or
use. Several techniques have been used to recover egg-albumin to allow the spermatozoa to migrate into the
spermatozoa from the tail of epididymis namely cutting, medium and then be recovered.

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Volume 7, Issue 10, October – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
D. Spermatozoa viability
The evaluation of motility and vigor consisted on the
deposition of a drop of semen in sodium chloride solution
(0.9%) for dilution, with subsequent removal of a sample and
depositing it on a pre-heated blade (37°C). This was inferred
using a contrast microscope, under a 10 and 40x objective,
and the result was issued as a subjective percentage,
according to the criterion adapted from Derivaux (1976), and
Baracaldo (2007). Vigor was rated using a scale from 0 to 5.

Morphology parameters were analyzed through

observation, under the microscope, of fixed smears with
methanol and stained with Giemsa, adapted from the FIG. 2. VALUES OF SPERMATOZOA VIGOR
technique described by Diaz and Arancibia (1970).
Percentages of abnormalities were obtained from 100 sperms Skimmed milk had a greater preservative effect
counted. compared to egg albumin and coconut water, with a
maximum motility preservative effect of about 44.33% on the
E. Statistical analysis first day and a minimum of 5% on the sixth day (148 hours)
Samples were chosen and randomly divided into 3 after the death of the bulls and sperm recovery. On the other
treatments, skimmed milk, egg albumin and coconut water. hand, it was observed that egg albumin similarly presented
For the statistical analysis of the data, the means and standard viable spermatozoa for the same period as milk, however with
deviations for the sperm parameters were obtained, using the relatively lower motility percentages and having presented a
analysis of variance (ANOVA), through the statistical maximum preservative effect of 34.33% on the first day and
program (SPSS), and using the Tukey test P<0.05 to assess a minimum of 3.30% on the sixth day. Coconut water had a
the significance of the differences of means between the three variation in sperm motility of 42.66% to 30.54% between the
tested preservatives. first and second day, showing total sperm inactivity from the
third day after sperm recovery. With regard to sperm vigor,
III. RESULTS an almost similar trend to motility was observed over the
period, with a preservative effect greater than 3 in the first
Conservation tests showed effect up to 6 days (148 day after recovery for the three treatments.
hours), with skimmed milk showing the best result in
preserving sperm motility and vigor, followed by egg Turning to abnormalities, defects in the tail were
albumin and finally coconut water. There were no significant predominant, showing a percentage variation of 12, 19 and
differences between three preservatives under refrigerated 29% in samples preserved in milk, egg albumin and coconut
conditions of 50C (p>0.05). The results of motility and vigor water, respectively, as illustrated below (Table 1).
are shown below in figure 1 and 2 respectively. Abnormalities in the head were more significant in milk
samples with 32% and the least in coconut water with 8%.
Intermediate part defects range from 6% to 19%.

Table I. Percentages of abnormalities

Treatment Type of Total of
abnormalities and abnormalities
location (%)
(%)
H I.P T
Skimmed 32 6 62 50
Milk
Coconut 8 13 79 50
water
Fig. 1. Percentages of spermatozoa motility Egg- 14 19 67 47
albumin
H: Head, I.P: Intermediate Peace and T: Tail

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Volume 7, Issue 10, October – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
IV. DISCUSSION (Dziekońska et al., 2020). Based on the location, this can be
divided into primary (abnormalities in the spermatozoa’s
The results found in this study can be explained head), secondary (abnormalities in the spermatozoa’s mid
according to Dziekońska et al. (2020) who mention that the piece) and tertiary (abnormalities in the spermatozoa’s tail)
tail of epididymis provide an environment which allow to (Corbet et al., 2012). In this study, the majority of
store gametes in physiological situations and, thus, viable abnormalities were found in the tail. This can be a result of
spermatozoa from this portion can be recovered after the the refrigerating process which, according to Souza et al.
death of the males. In addition, Martinez-Pastor et al. (2006), (2017) abnormalities in this portion increases significantly
O’Hara et al. (2010), Monteiro et al. (2013) and Nichi et al. after cooling as well as the total pathologies. Similar results
(2017) consider that refrigerating epididymis at 5°C, before of the morphological evaluation of the spermatozoa
sperm recovery, increases the sperm viability period by recovered from the epididymis were found by Costa et al.
slowing down the metabolic and degeneration rate. (2011) in which this problem was attributed to the immaturity
of the material recovered directly from the epididymis
Skimmed milk showed higher rates of preservative meaning that possibly the spermatozoa were recovered from
effects in comparison to the remaining two. This may be young bovine males .
regarded to its protein fraction and abundance of
carbohydrates (lactose) that can be used as source of energy. In conclusion, the results of this study demonstrated that
Besides that, milk contains casein that enhances the kinetic floating is a very viable technique for the recovery of
activity of sperm (Martins, 2018). Some preservative effects epididymal spermatozoa in cattle after death and that
of skimmed milk in bovine semen was also studied by skimmed milk, egg albumin and coconut water have natural
Thacker and Aimqlist (1958) who found that it is possible to preservative effects that may be helpful to reduce the loss of
extend sperms lifetime up to 16 days using skimmed milk. germplasm in Mozambique.

Egg albumin showed an ability to preserve the ACKNOWLEDGMENT

spermatozoa viability relatively lower than skimmed milk
however higher than coconut water. Albumin is a viscose This research did not receive any specific grant from
media and this may have led to much energy expenditure funding agencies either public, commercial, or non-for-profit
during the sperm migration from epididymis to the respective agencies.
media resulting in reduced effects agreeing with Ratnawati et
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