Tropical Animal Health and Production (2024) 56:200

https://doi.org/10.1007/s11250-024-04057-0

REGULAR ARTICLES

Dietary supplementing South African indigenous rams with flaxseed

oil and ascorbic acid improves cryopreserved semen quality and in
vitro fertility
Jabulani Nkululeko Ngcobo1 · Tshimangadzo Lucky Nedambale1 · Khathutshelo Agree Nephawe1 ·
Sindisiwe Mbali Sithole1,2 · Tlou Caswell Chokoe3 · Fhulufhelo Vincent Ramukhithi1

Received: 28 December 2023 / Accepted: 20 June 2024

© The Author(s) 2024

Abstract
The purpose of this study was to evaluate how ascorbic acid with dietary flaxseed oil affects the quality and fertility of
cryopreserved ram sperm in South African indigenous rams. Treatment diets were supplemented 60 days before semen
collection to afford proper spermatogenesis, adaptation to the feed formulated and fed throughout the study. Semen was
collected with the use of artificial vagina following dietary supplementation with five treatment diets (neg. cont. – nega-
tive control, pos. cont. – positive control, FLO – 5% Flaxseed oil, ASA – 4% Ascorbic acid, and FLO + ASA). Semen
was then extended using tris-based extender and cryopreserved using the programmable freezer (CBS Freezer 2100 series,
Laboratory consumables & chemical suppliers, America). Ovaries were collected from a neighbouring slaughter house
and conveyed to the lab in 0.9% saline at 37 °C. Data (sperm parameters and in vitro fertility) was then exposed to the
GLM (General Linear Model) in Minitab 17. Pearson’s correlation coefficient was utilized to investigate the relationship
between cryopreserved sperm quality and in vitro fertility. The student Least Significant Difference Test was used to
separate the treatment means, and differences were accepted when the p-value was less than 0.05. The FLO + ASA group
had higher (p < 0.05) progressive (36.33 ± 1.87), total (88.24 ± 2.24), rapid motility (27.52 ± 1.74), intact plasma mem-
brane (75.67 ± 2.08), total fertilization (65.98 ± 7.39), and total cleavage (66.19 ± 6.50) when compared to other treatment
groups. Total fertilization rate had a medium significant (p < 0.001) medium correlation with the progressive motility
(r2 = 0.435), total motility (r2 = 0.447) and rapid motility (r2 = 0.409). In conclusion, dietary flaxseed and ascorbic acid
(FLO + ASA) improves cryopreserved semen quality, in vitro fertilization rate, and the total cleavage rate. Noteworthy,
the progressive, total and rapid motility play a crucial in the in vitro fertilization rate.

Keywords Conservation · Docosahexaenoic acid · Long-chain Polyunsaturated fatty acids · Zulu · BaPedi · Namaqua
Afrikaner

Introduction

Food insecurity remain a challenge in South Africa and

Jabulani Nkululeko Ngcobo
jabulaninkululeko@gmail.com
1
Department of Animal Sciences, Tshwane University of
Technology, Private Bag X680, Pretoria 0001, South Africa
2
Agricultural Research Council, Germplasm, Conservation,
Reproductive Biotechnologies, Private Bag X02, Irene
0062, South Africa
3
Department of Agriculture, Land Reform and Rural
Development, Directorate Farm Animal Genetic Resource,
Private Bag X250, Pretoria 0001, South Africa
globally (FAO 2022). According to Dlamini et al. (2023),
about 20.4% of South African household are food insecure
with the low socio-economic house being most affected.
Domestic animals provide alternative source of protein to
feed human beings (Kunene et al. 2009). The indigenous
sheep of South Africa (Zulu, Bapedi, and Namaqua Afri-
kaner) are now threatened with extinction (Mazzera 2024).
Their animal genetic materials can be conserved through
cryopreserving semen for future use purposes and in vitro

13
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200 Page 2 of 12
embryo production and ensuing embryo transfer (Mara et al.
2013; FAO 2014; Ngcobo et al. 2022).
Sperm cryopreservation is the most vital advanced repro-
ductive biotechnologies in ruminants’ artificial insemina-
tion, because it allows the transport of semen from research
stations to farms and allow the storage of semen for longer
period of time (Leroy et al. 2019). Cryopreservation pro-
cedures and freezing mediums has been developed how-
ever, the frozen thawed small ruminants’ spermatozoa are
still much lower than that of liquid stored (Salamon and
Maxwell 2000). It is well known that only ± 60% of sperm
cells remain viable and motile after cryopreservation (Lv
et al. 2019) and only ± 30% remain biological functional
(Salamon and Maxwell 2000). Furthermore, previous stud-
ies reported that cryopreserved sperm survival is not only
dependent on species but also related to the individual and
some environmental factors such as management and nutri-
tional effects (Khan et al. 2021). For that reason, numer-
ous plant extract has been added to ram semen diluent to
improve cryopreserved semen quality (Ren et al. 2021).
Moreover, other studies have tried to improve cryopre-
served ram semen through supplementing long chain poly-
unsaturated fatty acid sources such as fish oil successfully
(Taghilou et al. 2017). However, supplementing fish oil
might increase human-animals food competition hence can
lead to the failure to feed highly growing population as pro-
jected by (FAO 2022). Therefore, this necessitate the need
to harness alternative source of long-chain polyunsaturated
fatty acids (LCPUFAs) particularly the plant-based precur-
sors such as the flaxseed oil.
Flaxseed oil has a high concentration of alpha-linolenic
acid (58%) that is broken down de novo to generate doco-
sahexaenoic acid, which is essential for testicular function
(Francois et al. 2003; Zanussi et al. 2019; Ngcobo et al.
2021). Docosahexaenoic acid (DHA) and eicosapentaenoic
(EPA) synthesised de novo through supplementing flaxseed
oil alter the sperm cell membrane structure, resulting in a
fluid sperm membrane and participation in cell response
mediated by protein (Tang et al. 2019). Nevertheless, sup-
plementing with flaxseed oil require a natural antioxidant to
balance the influence. Ascorbic acid, is a naturally occur-
ring antioxidant that has been shown to boost sperm qual-
ity and eliminate free radicals (Attia et al. 2020) when fed
with the long chain polyunsaturated fatty acid processors
(Singh et al. 2021). Despite intensive work done to inves-
tigate influence of flaxseed oil on fresh semen quality from
rams (Ngcobo et al. 2022, 2023), there are few (if any) stud-
ies that evaluated the influence of dietary inclusion of flax-
seed oil and ascorbic acid on cryopreserved sperm quality
and in vitro fertility. Therefore, determining how flaxseed
oil and ascorbic acid affects cryopreserved sperm quality,
and in vitro fertilization would pave the way for improved
13
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Tropical Animal Health and Production (2024) 56:200
sperm survival and fertility. Thus, the goal of this study was
to determine the effect of flaxseed oil and ascorbic acid on
cryopreserved sperm quality, and in vitro fertilization.
Materials and methods
Ethic statement
The study procedures were authorized by the Agricultural
Research Council (APAEC 2019/33) and the Tshwane Uni-
versity of Technology Animal Research Ethics Committee
(AREC2020/05/001).
Experimental site
The current study was carried out at the South African Agri-
cultural Research Council (ARC), Irene, Animal Production
(ARC, Irene). This region is located between 25o53’59.6”
South latitude and 28o12’51.6” East longitude. In this
region, sheep are reproductive active during late autumn to
winter season normally between March – May (Malejane
et al. 2014). Animals were kept in a same environmental
condition with free access to water but no access to the grass
pasture throughout the study.
Experimental animals
Study rams were housed in 5 trial pens as per treatment diets
to limit stress while allowing socialization among rams.
In this study, 22 matured indigenous South African rams
(age = 6 years, average body weight = 64.4 ± 1.6) were used.
They were then fed in the morning every day with water
provided ad-libitum. Twenty-two study rams were randomly
allocated 5 diets (neg. cont.), pos. cont., FLO, ASA and
FLO + ASA). The neg. cont. diet had 4 rams pos. cont. had
4 rams FLO had 5 rams ASA had 4 rams and FLO + ASA
diet had 5 rams. The treatment diets were formulated as
described by the National Research Council, 1985. Follow-
ing the formulation of treatment diets, FLO diet was top
mixed with 5% flaxseed oil, ASA diet was mixed with 4%
ascorbic acid and FLO + ASA diet was mixed with 5% flax-
seed oil and 4% ascorbic acid. Moreover, negative control
was the diet in use at the Agricultural Research Council,
Irene made of eragrostis curvular hay and 200 gram of pel-
lets per ram/day whereas, the positive control was the diet
formulated according to NRC (1985) without flaxseed oil
and ascorbic acid. Thereafter, the formulated diets were then
sampled and taken to South African National Accreditation
System (SANAS) accredited laboratory for feed composi-
tion analysis can be found in Table 1. Feed ingredients used
Tropical Animal Health and Production (2024) 56:200
Table 1 Proximate analysis of Proximate analysis Neg. Cont.
the treatment diets (Ngcobo et
Dry matter 88.23
al. 2023)
Ash 5.32
Crude Protein 10.73
Either Extract 2.03
Crude Fiber 29.01
in this study included Eragrostis curvular hay, soya bean,
bran, limestone and P12 bio mineral.
NB – neg. cont. (standard diet), pos. cont. (basal diet),
FLO (basal diet top dressed with 5% flaxseed oil), ASA
(basal diet top dressed with 4% Ascorbic Acid), FLO + ASA
(basal diet top dressed with both 5% flaxseed oil + 4%
Ascorbic Acid).
Experimental design
Semen collection and evaluation
Semen was collected by the artificial vagina following a 3
weeks training period and 60 days of supplementing with
five study diets during breeding season (March to June,
2022). Semen was collected twice a week to maintain 2
days resting period between collection for 3 months (March,
April, May and June) giving us a total of 704 ejaculates
collected from all rams. Computer-aided sperm analysis
(CASA), Sperm Class Analyzer® system (SCA) 5.0 ver-
sion (Microptic, Barcelona, Spain) at a magnification of 10x
(Nikon, Japan) and mounted with a warm plate (37 °C) was
used to assess sperm motility parameters following semen
cryopreservation, before in vitro fertilization. Sperm cell
parameters evaluated using CASA included total motil-
ity, non-progressive motility, progressive motility, rapid,
medium and slow motility, curvilinear velocity, average
path velocity, straight line velocity, linearity, straight-line
and wobble. Concoction of sperm and tris-swim up was
pipetted to the warmed microscope slide (76 × 26 × 1 mm,
Germany) and gently enclosed with cover slip (22 × 22 mm,
Germany) for sperm motility parameters. A total of four
fields containing ± 200 sperm were randomly selected per
field and analysed using Computer-Aided sperm analysis
(CASA). Sperm plasma membrane was evaluated using the
hypo-osmotic swelling test (HOST) as reported by (Chatiza
et al. 2018) with a few modifications. In brief, 100µL cryo-
preserved semen was mixed with 500µL HOST (sodium
citrate = 0.3675, fructose = 0.6755 in 50 ml Falcon tube)
and incubated at 37 °C for 30 min. Following 30 min of co-
incubation, 10µL of the concoction was pipetted and gently
distributed on the microscope slide, which was then exam-
ined under the microscope as follows: 1 - intact sperm cells
with a curved tail, 2 - non-intact/damaged sperm cells with
a straight tail.
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Page 3 of 12 200
Pos. Cont. FLO ASA FLO + ASA
88.93 92.20 90.42 93.52
12.36 5.00 5.49 4.75
10.36 10.74 10.29 10.59
2.16 19.79 1.70 24.29
21.71 25.90 28.71 20.95
Sperm morphology and viability was assessed follow-
ing cryopreservation, before in vitro fertilization. Cryopre-
served semen samples were mixed with the eosin-nigrosin
(5% Eosin and 10% nigrosin) stain (Ondersterpoort Faculty
of Veterinary Science’ Pharmacy, South Africa) at a ratio of
5µL:20µL. The concoction was distributed on the micro-
scope slide gently and dried at a room temperature for 24 h
before evaluation. After 24 h, immersion oil was smeared
and the total of 200 sperm cells were counted under the
Olympus microscope (Olympus microscope, BX51, Japan)
magnificent (UPlan FLN, 100x/1.30 011 /0.17 / FN26.5).
Sperm abnormalities were classified as primary, secondary
and tertiary abnormalities. Primary abnormalities are the
abnormalities occurring during spermatogenesis including
double head and double tail. Secondary abnormalities are
the abnormalities occurring during ejaculation and these
includes: proximal droplets and distal droplets. Tertiary
abnormalities are those occurring due to the in vitro sperm
handling and includes: coiled tail and bent tail.
Experiment 1: semen cryopreservation
Semen samples were then evaluated for sperm motility and
only semen samples with between 80 and 90% total motil-
ity was cryopreserved. Tris-egg yolk-based extender was
used to extend semen samples. The composition of the Tris-
egg yolk-based extender chemical composition were: tris,
2.11 g; citric acid, 0.68; monohydrate glucose, 0.5; genta-
mycin sulphate, 0.05; egg yolk 10mL and deionized water
of 40mL (Salamon and Maxwell 2000). Glycerol (9mL) was
added on the second fraction. After four hours of equilibra-
tion at 5˚C, semen samples were loaded to 0,25mL poly-
vinyl alcohol semen straws using different colours as per
treatment diets.
Programmable freezer (CBS Freezer 2100 series, Labo-
ratory consumables & chemical suppliers, America) was
used to cryopreserve semen with the following rate settings:
step 1 – cooling rate 0.00 (C/min), demand temperature 5˚C
and the holding time of 0.00 min, step 2: cooling rate of
0.08˚C/min, demand temperature of 4˚C, holding time of
5 min, step 3: cooling rate 6˚C/min, demand temperature
of -130˚C with the holding time of 10 min. Following cryo-
preservation with the programmable freezer (CBS Freezer
2100 series, Laboratory consumables & chemical suppliers,
America), semen straws were dipped to the Styrofoam box
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(Plastilion packaging, South Africa) with liquid nitrogen.
Semen straws were then loaded in the cannisters and kept for
6 months. On the day of in vitro fertilization, semen straws
were thawed using warm water (37 ˚C) for 1 min and evalu-
ated using CASA before use either for in vitro fertilization.
Experiment 2: fertility of cryopreserved semen
Sheep ovary collection Sheep ovaries were taken within
2 h after slaughter from a local slaughterhouse (Cavalier
Foods (Pty) Ltd), Gauteng Province and conveyed to the
laboratory in a thermos flask containing 0.9% buffered
saline water (Adcock Ingram critical care, SA) at 37 °C. Up
on arrival at the laboratory ovaries were rinsed with clean
saline water to eliminate blood contamination from donor
sheep and sprayed with 75% ethanol absolute (Laboratory
Consumables and Chemicals supply cc, SA).
Oocytes retrieval and grading Ovaries were selected and
put on a petri dish (Falcon 1008, USA) with modified Dul-
becco’s phosphate buffered saline (mDPBS), processed
through slicing method by chopping ovaries into tiny pieces
using a surgical blade (Hi-Careint, Japan), and transferred
into a 50 mL falcon tube (Plastpro Scientific, SA) contain-
ing wash medium® (Bioscience, UK). Follicular fluid was
transferred into a graded petri dish (Carbi, USA), and COCs
were selected from the mixture using a microscope (Olym-
pus BX71, Philippines) at a magnification of 10x/H22.
The oocytes were categorised as grade A, B, or C, with
grade A oocytes containing several entire layers of cumulus
cells and uniform cytoplasm. Grade B oocytes had partial
layers of cumulus cells and homogenous cytoplasm, while
Grade C oocytes had no cumulus cells. Oocytes retrieved in
grades A, B, and C was counted, however only grade A was
used for In Vitro Maturation (IVM), In Vitro Fertilization
(IVF) and In Vitro Culture (IVC).
In vitro maturation of oocytes After collecting, processing,
and grading all oocytes, a total of 500 L Brackett and Oli-
fant (BO) wash medium® (Bioscience, UK) washed grade
A oocytes three times. These oocytes were in vitro matured
in 4-well pans with 500 L BO-IVM® (Bioscience, UK)
enriched with low glucose, gonadotrophic hormones, and
gentamycin. Following that, the oocytes were incubated for
24 h at 38.5 OC with 5% CO2, 5% oxygen (O2), and 100%
humidity.
Polar body evaluation After 24 h, a portion (25%) of
matured oocytes were out of the incubator and vortexed for
13
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Tropical Animal Health and Production (2024) 56:200
3 min in 200µL while the remaining oocytes (75%) were
kept for IVF purposes. They were then placed on a petri dish
with 3 mL of BO wash media for polar body extrusion. The
Oosight Imaging System (Hamilton Thorne) connected to
the microscope Olympus 1 × 71 (New York microscope Co,
USA) at 20x/0.45 Rc2 magnification was used to examine
the vortexed oocytes for polar body extrusion between the
zona pellucida and the cytoplasmic space.
Preparation of matured oocytes for in vitro fertiliza-
tion Oocytes matured in BO IVM® media were washed
five times in 50µL BO-IVF medium (IVF Bioscience, UK).
These oocytes were then placed into 100µL fertilization
micro drops coated with 3 mL mineral oil.
Thawing of frozen semen straw and sperm capacitation In
vitro fertilization was achieved by utilizing cryopreserved
sperm collected from the experimental rams according to
their treatment diets (neg. cont., pos. cont., FLO, ASA, and
FLO + ASA). (Ngcobo et al. 2023). For thawing of semen,
the frozen straws from each diet were held in the air for ten
seconds before being dropped into 37 °C water for a min.
The straws were dried and sliced on both sealed ends, and
the contents were emptied into 15 mL falcon® tubes (Nest
Biotechnology Co, China). Prior fertilization, sperm (5µL)
distributed in a warm microscope slide (Labocare, UK) to
examine the sperm motility parameters under the CASA
system. The BO semen prep® media was used to select
sperm (IVF Bioscience, UK). The contents of the frozen
thawed straws were diluted with 4 mL of prewarmed semen
preparation medium in 15 mL tubes and centrifuged at
1500 g force for 5 min. Following centrifugation, the super-
natant was removed, and sperm suspension remained in a
4 mL tube; then, fresh warmed BO semen prep® medium
was added, and the combination was resuspended and cen-
trifuged. Following the second centrifugation, the super-
natant was removed until the same volume of 350–700µL
sperm suspension remained; the pellet in this volume with a
concentration of 2.0 × 106/mL was diluted with semen prep
medium, suspended, and utilized for IVF.
In vitro fertilization of matured oocytes Oocytes remained
from polar body evaluation were fertilized with their respec-
tive dietary cryopreserved semen. Twenty-five (25%) of
these oocytes were incubated for 6 h for pronucleus evalu-
ation, while the remaining 50 oocytes were incubated for
Tropical Animal Health and Production (2024) 56:200 Page 5 of 12 200

Table 2 Effect of treatment diets on cryopreserved semen quality following dietary flaxseed oil and ascorbic acid
Parameters Neg. Cont. Pos. Cont. FLO ASA FLO + ASA
Progressive motility 19.97 ± 1.87c 19.80 ± 1.87c 28.19 ± 1.87b 27.02 ± 1.87b 36.33 ± 1.87a
None-progressive motility 29.36 ± 2.24c 44.20 ± 2.24b 52.51 ± 2.24a 52.32 ± 2.24a 51.91 ± 2.24a
Total motility 49.33 ± 2.24d 64.00 ± 2.24c 80.70 ± 2.24b 79.34 ± 2.24b 88.24 ± 2.24a
Rapid motility 14.07 ± 1.74bc 13.20 ± 1.74c 18.92 ± 1.74b 17.73 ± 1.74bc 27.52 ± 1.74a
Medium motility 7.73 ± 1.05b 9.94 ± 1.05b 14.10 ± 1.05a 14.93 ± 1.05a 16.16 ± 1.05a
Slow motility 27.53 ± 2.12c 40.86 ± 2.12b 47.68 ± 2.12a 46.68 ± 2.12ab 44.54 ± 2.12ab
Sperm plasma membrane integrity (%)
Intact Sperm 52.33 ± 2.08c 58.13 ± 2.08c 70.87 ± 2.08ab 69.67 ± 2.08b 75.67 ± 2.08a
Sperm viability (%)
Live Sperm 65.00 ± 1.37b 60.93 ± 1.37c 74.20 ± 1.37a 72.27 ± 1.37a 75.73 ± 1.37a
Sperm abnormalities (%)
Primary 13.00 ± 0.96a 7.07 ± 0.96b 4.20 ± 0.96c 4.27 ± 0.96c 4.27 ± 0.96c
a ab b a
Secondary 7.13 ± 0.89 5.47 ± 0.89 3.67 ± 0.89 6.47 ± 0.89 3.47 ± 0.89b
b a ab b
Tertiary 14.80 ± 1.85 20.13 ± 1.85 16.93 ± 1.85 13.80 ± 1.85 17.47 ± 1.85ab
a, b, c
Means with different superscripts within the raw, differ significantly (p < 0.5). NB – neg. cont. (standard diet), pos. cont. (basal diet), FLO
(basal diet top dressed with 5% flaxseed oil), ASA (basal diet top dressed with 4% Ascorbic Acid), FLO + ASA (basal diet top dressed with
both 5% flaxseed oil + 4% Ascorbic Acid)

18 h at 38.5 OC with 5% CO2, 5% O2, and 100% humidity
for IVC purposes.
Preparation of presumptive zygotes for staining and pro-
nucleus evaluation Following 6 h of IVF, presumptive
zygotes were extracted from fertilization drops and cumulus
cells were extracted by vortexing in 200 L of pre-warmed
wash media (Bioscience, UK) for one min and 30 s. The
zygotes were then washed in wash medium before being
stained with a Hoechst solution.
IVC® medium (Bioscience, UK) drops and moved into the
new 100µL BO IVC® drops of the same medium that had
been coated with mineral oil. These were then cultured for
48 h in modular chamber with 5% O2 and 5% CO2 mixed
gas added for a min and incubated for 48 h. After 48 h of
culture, the sperm-exposed presumptive zygotes were
examined for cleavage rate (day two of IVC).
Statistical analysis

Four drops of Petroleum jelly (Vaseline, Unilever South
Africa) were placed in a square pattern around a tiny slide.
Presumptive zygotes were moved to a glass slide. Hoechst
solution (2–10µL) (stock 2) of was applied to the sides of
the cover slip up until the entire surface underneath the slip
was coated. This was done with caution to avoid washing
the presumptive zygotes out of the slide. A cover slip (Lab-
ocare, UK) was gently pushed over the microscopic slide
until reaching the presumptive zygotes drop. The cover
slip’s ends were immediately sealed with colourless nail
polish. Prepared slides dried for 2 h in a dark area before
examination using an inverted microscope Olympus 1 × 71
(New York microscopy Co, USA) at 20x/0.45 Rc2 magnifi-
cation with a UV filter. After 2 h, the slides were examined
for pronucleus status.
In vitro culture of presumptive zygotes For IVC pur-
poses, presumptive zygotes were transferred from fertiliza-
tion drops into pre-warmed wash medium®, and vortexed
for 1 min 10 s for removal of cumulus cells. Presumptive
zygotes were washed five times in 50µL of pre-warmed BO
Data (sperm parameters and in vitro fertility parameters)
was exposed to a General Linear Model (GLM) in Minitab
17®. Pearson’s correlation coefficient was utilized to cor-
relate cryopreserved sperm quality and in vitro fertility
parameters. The student Least Significant Difference Test
was used to separate the treatment means, and differences
were accepted when the p-value was less than 0.05.
Results
Semen cryopreservation
The current study investigated the effect of flaxseed oil and
Ascorbic acid on the cryopreserved semen quality and fer-
tility and the findings are shown in Table 2. The progres-
sive motility was higher in FLO + ASA treated group than
neg. cont., pos. cont., FLO and ASA. The total motility was
higher (p < 0.05) in the FLO + ASA than the neg. cont., pos.
cont., FLO and ASA. Static motility was higher in the neg.

13
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 200 Page 6 of 12
cont., than that in the FLO + ASA, ASA, FLO and the pos.
cont. groups.
Rapid motility was higher (p < 0.05) in the FLO + ASA
groups when compared with that of ASA, FLO, positive
and the neg. cont. On the other hand, Medium motility in
the FA + ASA and ASA were higher (p < 0.05) than that
of neg. cont. and the pos. cont. Slow motility was higher
in the FLO + ASA, ASA, FLO than the neg. cont. group.
The dietary treatment effect was also investigated through
evaluating the sperm plasma membrane, viability and sperm
abnormalities in cryopreserved semen following dietary
supplementation of flaxseed oil and Ascorbic acid and the
results are obtainable in Table 2. The effect of treatment
diets (neg. cont., pos. cont., FLO, ASA and FLO + ASA)
on sperm plasma membrane, viability and abnormali-
ties were also evaluated and the results are obtainable in
Table 2. There was a higher (p < 0.05) intact sperm cells in
FLO + ASA and FLO (70.87 ± 2.08) than that of the neg.
cont. and the pos. cont. Intact sperm cells in ASA could not
differ (p > 0.05) to FLO diet.
For sperm viability, there was a greater (p < 0.05) live
sperm cells following supplementing the FLO + ASA, ASA
and the FLO than that of the positive and the neg. cont.
Primary abnormalities could not differ (p > 0.05) among
FLO + ASA, ASA and FLO however, were lower than
that of the pos. cont. and the neg. cont. Secondary sperm
abnormalities were higher in the ASA, neg. cont. and the
pos. cont. than that from FLO + ASA and the FLO. Tertiary
sperm abnormalities were higher in the pos. cont., than the
neg. cont., FLO, ASA and FLO + ASA.
Fig. 1 Illustrate in vitro culture (A-C – in vitro culture), sperm viability (D – live sperm, E – dead sperm), some sperm abnormalities (F – detached
sperm/tailless sperm, G – bent tail and H – coiled tail) and I - Computer-Aided Sperm analyser
13
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Tropical Animal Health and Production (2024) 56:200
Fertility of cryopreserved semen
The pronucleus evaluation following the use of cryopre-
served semen (Fig. 1) was conducted and the results are
obtainable in Table 3. There were higher (p < 0.05) none PN
oocytes in the neg. cont., pos. cont., FLO, and ASA groups
than that of FLO + ASA group. No differences observed for
the 1 PN, 2PN and > 2PN among all treatment groups (neg.
cont., pos. cont., ASA, FLO and FLO + ASA). However, the
total fertilization was higher (p < 0.05) in the FLO + ASA
group than the neg. cont., pos. cont. and ASA treated groups.
The dietary effects (neg. cont., pos. cont., FLO, ASA and
FLO + ASA) on the cleavage rate (Fig. 1) was investigated
and the results can be found in Table 3. The number of pre-
sumptive zygotes cleaved was the same for all the treat-
ment diets. The FLO recorded the lowest 1cell percentage
when compared to neg. cont., pos. cont., and ASA however
not different from FLO + ASA. The FLO and FLO + ASA
recorded a significant higher percentage of the presump-
tive zygotes (2-4cells) when compared to neg. cont., pos.
cont., and ASA. No significant difference recorded on the
presumptive zygotes with 6-8cell on the pos. cont., FLO,
and ASA nevertheless none were recorded for the neg. cont.
and the FLO + ASA. Noteworthy, the FLO (67.14%) and the
FLO + ASA recorded greater total cleavage rate in compari-
son with the neg. cont., pos. cont., and ASA.
The relationship between cryopreserved sperm param-
eters and the IVF, IVC and the conception rate were cor-
related using the Pearson’s correlation coefficient and the
results are obtainable in Table 4. Two pronucleus had a
medium relationship with progressive motility (r2 = 0.331),
non-progressive motility (r2 = 0.303), total motility
Tropical Animal Health and Production (2024) 56:200 Page 7 of 12 200

Table 3 Depicts the dietary effect on in vitro fertilization

Parameters Neg. Cont. Pos. Cont. FLO ASA FLO + ASA
In vitro fertilization rate (%)
No. of IVF oocytes 8.33 ± 0.66ab 9.17 ± 0.70ab 7.36 ± 0.60b 9.06 ± 0.77ab 9.56 ± 0.77a
a a ab a
None PN% 59.61 ± 6.43 62.62 ± 6.82 47.47 ± 5.80 56.01 ± 6.43 34.02 ± 6.82b
a a a a
1 PN% 21.83 ± 4.28 24.60 ± 4.54 28.59 ± 3.86 20.93 ± 4.28 34.58 ± 5.02a
a a a a
2 PN% 7.08 ± 5.07 7.78 ± 5.38 14.50 ± 4.57 18.44 ± 5.07 23.15 ± 5.95a
a a a a
> 2 PN% 13.15 ± 4.14 7.38 ± 4.39 8.39 ± 3.73 4.63 ± 4.14 8.25 ± 4.86a
b b ab b
TF% 42.06 ± 6.30 39.76 ± 6.68 51.49 ± 5.68 43.99 ± 6.30 65.98 ± 7.39a
In vitro cleavage rate (%)
Lysed 0 0 0 0 0
Oocytes cleaved 8.67 ± 0.43a 9.06 ± 0.40a 9.50 ± 0.43a 9.50 ± 0.43a 9.50 ± 0.43a
ab a d bc
1Cell% 71.62 ± 6.50 75.60 ± 6.13 32.86 ± 6.50 52.82 ± 6.50 33.81 ± 6.50cd
c c a b
2-4Cell% 28.38 ± 5.55 23.01 ± 5.24 63.81 ± 5.55 45.51 ± 5.55 66.19 ± 5.55a
6-8Cell% 0 a a a 0
1.39 ± 1.53 3.33 ± 1. ±63 1.67 ± 1.63
Total cleavage% 28.38 ± 6.50cd 24.40 ± 6.13d 67.14 ± 6.50a 47.18 ± 6.50bc 66.19 ± 6.50ab
a, b
Means with different superscripts within the raw differ significantly (p < 0.05). neg. cont. (standard diet), pos. cont. (basal diet), FLO (basal
diet top dressed with 5% flaxseed oil), ASA (basal diet top dressed with 4% Ascorbic Acid), FLO + ASA (basal diet top dressed with both 5%
flaxseed oil + 4% ascorbic acid). IVF – In vitro fertilization, 1PN – 1 Pronucleus, 2PN – 2 Pronucleus, >2PN – Polyspermy, TF – Total Fertil-
ization

(r2 = 0.418) and medium motility (r2 = 0.331). A weak rela-
tionship was observed between 2 pronucleus and rapid
motility (r2 = 0.292). Total fertilization rate had a medium
significant (p < 0.001) medium correlation with the pro-
gressive motility (r2 = 0.435), total motility (r2 = 0.447) and
rapid motility (r2 = 0.409). For in vitro culture, there was a
significantly (p < 0.001) strong relationship between 2 and 4
cell and progressive motility (r2 = 0.558) and rapid motility
(r2 = 0.594) whereas, 2–4 cell had a significantly (p < 0.001)
medium relationship with the non-progressive motility and
the weaker relationship with slow motility (r2 = 0.358). The
total in vitro culture rate had a significantly (p < 0.001)
strong correlation with the total motility (r2 = 0.610) and
rapid motility (r2 = 0.539). Moreover, there was a substan-
tial (p 0.001) medium association between in vitro culture
and increasing motility (r2 = 0.422). The medium and slow
motility had a significantly (p < 0.001) weak correlation with
total in vitro culture (r2 = 0.302, r2 = 0.377), respectively.
Conception rate was not significantly (p > 0.05) correlated
to any of the sperm cell parameters following cryopreserved
processes.
Discussion
Semen cryopreservation
The objective of the study was to investigate the dietary
effects of flaxseed oil and Ascorbic acid on the quality
and fertility of cryopreserved sperm. The dietary supple-
mentation with flaxseed oil in South African rams’ diets
can improve fresh semen quality during breeding season
(Ngcobo et al. 2023) and throughout the years (Ngcobo et
al. 2023). As a results, dietary inclusion of dietary omega
n-3 sources to improve reproduction performance in sheep
is of a great interest to implement assisted reproductive tech-
nologies in the conservation program (Kargar et al. 2017).
Cryopreservation techniques increase plasma membrane
fluidity-permeability and reactive oxygen species genera-
tion (Peris-Frau et al. 2020). Moreover, molecular inves-
tigations demonstrated that sperm plasma membrane and
cholesterol levels changed after cryopreservation (Peris-
Frau et al. 2020).
Dietary flaxseed oil had an influence on cryopreserved
sperm quality where, flaxseed and Ascorbic acid supple-
mented group performed better than the negative and the
pos. cont. for progressive, non-progressive, total motil-
ity and rapid motility. Similar outcomes were observed by
(Kumar et al. 2021) in growing male lambs and in bucks out
of breeding season (Souza et al. 2019) rams out of breeding
season (Ngcobo et al. 2023) and in bulls (Moallem et al.
2015). Cryopreserved sperm quality improvement observed
in this study can be justified by the docosahexaenoic acid
since it is a main phospholipid coating the sperm plasm
membrane (Martínez-Soto et al. 2016), making up to 30%
of esterified fatty acids in the phospholipids and 73%of all
polyunsaturated fatty acids (Aké-Villanueva et al. 2019).
Flaxseed oil contain 58% alpha-linolenic acid, which is an
essential antioxidant known to improve animal health and
production (Khan 2015) and the general animal reproduc-
tive health (Perumal et al. 2019). Alpha-linolenic acid is a
docosahexaenoic acid (DHA) precursor synthesized with
the aid of ∆5 and ∆6-desaturase enzymes (Nandi et al. 2019;
Collodel et al. 2020). Apart from long-term conservation of

13
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 200

13
Page 8 of 12

Table 4 Pearson correlation between cryopreserved sperm parameters, in vitro fertilization, in vitro culture and the conception rate
Parameters 1PN 2PN > 2PN TF 1 Cell 2–4 Cell 6–8 Cell TC Progres- None- Total Rapid Medium Slow
sive pro- motility motility motility motility
motility gressive
motility
1PN 1.00
2PN 0.170NS 1.00
> 2PN −0.419** −0.371NS 1.00
TF 0.620*** 0.698*** −0.011NS 1.00
1 Cell −0.279* −0.358NS 0.080NS −0.440*** 1.00
2–4 Cell 0.321** 0.363** −0.124NS 0.449*** −0.987*** 1.00
6–8 Cell −0.133NS 0.101NS 0.216NS 0.110NS −0.435*** 0.283* 1.00
TC 0.279* 0.358** −0.080NS 0.440*** −1.000NS 0.987*** 0.435*** 1.00
Progressive motility 0.229NS 0.331* 0.008NS 0.435*** −0.508*** 0.558*** −0.044NS 0.508*** 1.00
Nnoe-progressive 0.258NS 0.303* −0.287* 0.259NS −0.422*** 0.414*** 0.201NS 0.422*** 0.132NS 1.00
motility
Total motility 0.324* 0.418** −0.205NS 0.447*** −0.610*** −0.610*** 0.091NS 0.610*** 0.690*** 0.809*** 1.00
Rapid motility 0.260NS 0.292* −0.431*** 0.409*** −0.539*** 0.594*** −0.111NS 0.539*** 0.948*** 0.096NS 0.632*** 1.00
Medium motility 0.126NS 0.331* −0.404*** 0.382*** −0.302* 0.329* −0.037NS 0.302** 0.660*** 0.416*** 0.695*** 0.439*** 1.00
Slow motility 0.237NS 0.259NS −0.206NS 0.189NS −0.377** 0.358** 0.244NS 0.377** 0.012NS 0.976*** 0.720*** −0.013NS 0.257NS 1.00
Conception rate 0.009NS −0.129NS 0.079NS −0.049NS 0.105NS −0.120NS 0.047NS −0.105NS −172NS 0.028NS −0.082NS −0.180NS −0.044 0.036NS
NS – Non-Significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001. PN – Pronucleus, TF – Total Fertilization, TC – Total Cleavage
Tropical Animal Health and Production

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(2024) 56:200
Tropical Animal Health and Production (2024) 56:200
sperm cells, cryopreservation reduces the cost of keeping
rams in the farm and the prevention of genetic drift (Bar-
bas and Mascarenhas 2009). Furthermore, cryopreserva-
tion simplifies worldwide sperm commerce and allows for
long-term preservation of superior sire sperm (Peña et al.
2011). Nevertheless, cryopreservation promotes numerous
cell damages including reduction in sperm membrane fluid-
ity, lipid, and protein organization (Fernandes et al. 2021).
However, ram sperm cells are sensitive to cryopreser-
vation processes in comparison to other domestic animals
(Curry 2000) due to higher sensitivity to low temperature
(Salamon and Maxwell 2000) and low cryotolerance hence,
less than 30% of sperm cells remain undamaged following
cryopreservation (Salamon and Maxwell 2000). This cryo-
damages are informed by oxidative, osmotic and thermal
stresses that are harmful to the sperm cells and can cause
poor conception rate (Tariq et al. 2015). In the current study,
when the influence of diet was introduced, FLO + ASA and
FLO had higher intact plasma membrane integrity when
compared to the negative and the pos. cont. following cryo-
preservation. It is generally established that cryopreservation
alters sperm cell motility, causes membrane modifications
affecting sperm capacitation and acrosome response, and
reduces sperm viability (Martin et al. 2004). Furthermore,
seminal plasma contains proteins that shield sperm against
cold shock damage and increase sperm viability (Pérez-Pé
et al. 2001). Among these proteins tripeptidyl-peptidase,
heat-shock protein 90 and chaperonin-containing t-complex
polypeptide 1 are the main protein regulating cryo-capaci-
tation, protect sperm against oxidative stress and stabilizes
sperm membrane through an efficient protein folding (Wang
et al. 2013; Rickard et al. 2015). These findings matched
those published by (Mandiki et al. 1998) in Texel, Suffolk,
and Ile-de-France rams. However, their results might be
justified by the season and the age of rams (Belkhiri et al.
2017). Noteworthy, the current study revealed that, rams fed
FA + ASA, ASA and FLO had significant good sperm viabil-
ity through higher live sperm cells and lower dead sperm
cells post-thawing. Sperm viability and plasma membrane
integrity has been reported as the strong indicators of sperm
functions (Peña et al. 2011). On the opposite hand, the cur-
rent study observed significant lower sperm abnormalities
in the dietary supplemented groups especially for primary
abnormalities.
Sperm abnormalities were then classified into primary,
secondary and tertiary sperm abnormalities (Ngcobo et al.
2023). Primary sperm abnormalities happen during sper-
matogenesis, secondary sperm abnormalities happen after
ejaculation, and tertiary abnormalities happen during invi-
tro sperm handling (Ngcobo et al. 2023). Sertoli cells in
the testicles are active in conversion of 18 and 20 carbon
omega n-3 into 22 carbon LCPUFAS (68) due to their high
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 9 of 12 200
concentration of desaturase mRNA and elongate enzymes
(Souza et al. 2019). Moreover, DHA provides essential
sperm membrane fluidity and mediate sperm cell response
to protein (Nandi et al. 2019) and supports the role of astro-
cyte, retina pigment epithelial cells and Sertoli cells in the
testicles (Sæther et al. 2007). High polyunsaturated fatty
acid and low antioxidant capacity in ram sperm cells lead to
the build-up of reactive oxygen species (ROS) during cryo-
preservation. Oxidative stress can be regulated by inclusion
of antioxidants during cryopreservation (Tariq et al. 2015).
Nevertheless, cryopreservation still produces high ROS,
osmotic stress, spermatic DNA damage, membrane destabi-
lization and dysfunction (Vichas et al. 2018).
Fertility of cryopreserved semen
Following evaluation of cryopreserved sperm quality after
dietary supplementation flaxseed oil and Ascorbic acid,
sperm cell was tested for in in vitro fertilization. Notewor-
thy, FLO + ASA had a highest total fertilization although
could not differ with that observed in FLO. Currently, in
our best knowledge there are less studies that evaluated the
influence of dietary flaxseed oil on in vitro fertilization rate.
Nevertheless, it is well known that only ± 60% of sperm
cells remain viable and motile after cryopreservation (Lv et
al. 2019) and only ± 30% remain biological functional (Sal-
amon and Maxwell 2000). Numerous domestic animals are
born following the use of assisted reproductive biotechnolo-
gies (Viana 2020.). Slaughtered ovary donor of a superior
genetic material can reduce the generation interval by ± 3–6
months (Amiridis and Cseh 2012) hence, cryo-gene bank
is a vital strategy in conservation of farm animal genetic
material (Leroy et al. 2019). However, generally there is a
decline in ovine embryo production with non-reported in
Africa (Viana 2020). As a result, the findings of this work
can make a significant contribution to the development of
effective cryo-gene banks for the preservation of embryos of
high genetic value and from endangered species.
In vitro culture result followed a similar trend to that
of in vitro fertilization. In brief, groups supplement with
FLO + ASA and FLO had the highest total cleavage in com-
parison to negative and the pos. cont. The neg. cont., and the
pos. cont. were both not supplemented with flaxseed oil and
it is known that low DHA in the sperm cell has been associ-
ated with the infertility (Moallem et al. 2015). Furthermore,
dietary supplementation of omega n-3 sources is proven to
increase sperm and subsequent fertilization rate motility
(Ahmad et al. 2019) thus the great interest in dietary inclu-
sion of dietary omega n-3 precursors on domestic livestock
diets (Díaz et al. 2017). Despite other numerous factors
affecting the efficient of in vitro embryo production, semen
13
 200 Page 10 of 12
quality (fresh or frozen) and the selection of semen has been
major factors (Falchi et al. 2018).
Sperm cells with defective DNA can fertilize, however
embryo development can be interrupted once the embryo
genome is active at the 4 or 8 cell stage due to improper pater-
nal gene transcription (Kumar and Naqvi 2014). Therefore,
there was a need to evaluate the conception rate following
dietary supplementation of flaxseed oil and Ascorbic acid.
Dietary (FLO and FLO + ASA) treated groups performed
better than other groups. Shahid et al. (2019), observed
similar results in chickens and suggested that flaxseed oil
enriched diets change the sperm PUFAs’ mostly during the
decreasing phase of annual reproductive period. Moreover,
in cows supplementing flaxseed oil Improved pregnancy
rate and reduced uterine PGF2α secretion and decreased
sensitivity of the corpus luteal to PGF2α during important
stage of embryonic development (Nazir et al. 2013).
In conclusion, dietary inclusion of FLO + ASA improve
frozen thawed sperm quality, total in vitro fertilization
and total cleavage rate. Therefore, the FLO can be used
to improve cryopreserved sperm quality. Furthermore, the
progressive, total and rapid motility play a crucial in the in
vitro fertilization rate. These results may pave a way to dis-
seminate superior genetic material and improve South Afri-
can indigenous sheep population through in vitro embryo
production.
Acknowledgements Acknowledgement goes to the National Research
Foundation - DAAD: German Academic Exchange Service scholar-
ship, Tshwane University of Technology (TUT), the Agricultural
Research Council (ARC), and the Agriculture Sector Education Train-
ing Authority (AgriSETA). South African Department of Agriculture,
Land Reform and Rural Development (DALRRD) - Farm Animal
Genetic Resource (FAnGR).
Authors contribution Jabulani Ngcobo conceptualized this work, Jab-
ulani Ngcobo and Sindisiwe Sithole collected data and formal analy-
sis; Fhulufhelo Ramukhithi provided laboratory resources; Jabulani
Ngcobo wrote the first draft of the article; Fhulufhelo Ramukhithi and
Khathutshelo Nephawe reviewed and edited the article; Tshimangadzo
Nedambale and Fhulufhelo Ramukhithi supervised the study, Caswel
Chokoe was responsible for fund acquisition. The published version of
the work has been reviewed and approved by all authors.
Funding Open access funding provided by Tshwane University of
Technology. The DARLD funded this study with project number
21.1.1/18/GR-02/API and the Agricultural Research Council with
grant number P02000172.
Open access funding provided by Tshwane University of Technology.
Data availability This study’s intellectual property is owned by TUT
and the ARC, Irene (ARC). However, the data is available from the
corresponding author when requested.
13
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Tropical Animal Health and Production (2024) 56:200
Declarations
Statement of Animal rights Animal ethics approval from this study was
obtained from the Agricultural Research Council, Irene Animal Re-
search ethics committee (APAEC 2019/33) and Tshwane University of
Technology Animal Research Ethics committee (AREC2020/05/001).
Conflict of interest The authors state that they have no conflicts of in-
terest.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format,
as long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate
if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons.
org/licenses/by/4.0/.
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