INTRODUCTION
High male fertility is crucial in the ruminant industry for producing quality semen essential for effective artificial insemination, which directly impacts farm profitability through improved conception rates and increased offspring per herd Semen quality is influenced by various factors, including genetics, management practices, environment, and nutrition Fatty acids are vital energy sources and key components of cellular structure and function Notably, polyunsaturated fatty acids (PUFAs) like Linoleic acid (LA), Alpha-Linolenic acid (ALA), Eicosapentaenoic acid (EPA), and Docosahexaenoic acid (DHA) can specifically target reproductive tissues, potentially enhancing reproductive function and fertility.
Mammalian sperm is characterized by a high proportion of polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA), with variations across species The lipid composition of sperm membranes is crucial for specific functions, as it facilitates the formation of microdomains with distinct fluidity and permeability, essential for successful oocyte fusion Additionally, the sperm plasma membrane's lipid makeup significantly influences mobility, cold sensitivity, viability, and membrane integrity PUFAs are vital for maintaining membrane fluidity and reducing lipid peroxidation risk, with n-3 and n-6 PUFAs constituting 30-50% of the total fatty acids in mammalian sperm membranes, thereby regulating fluidity and acrosome responsiveness Research has shown that increasing n-3 and n-6 PUFAs in pig sperm membranes through dietary adjustments can enhance reproductive activity.
Research indicates that the inclusion of polyunsaturated fatty acids (PUFA) in the diets of various animals, including bovines, goats, and rams, enhances sperm quality by altering the fatty acid composition of sperm membranes Studies by Khoshvaght et al (2015), Moallem et al (2015), Adibmoradi et al (2012), and Samadian et al (2010) support this finding, showing improved characteristics in both fresh and post-thaw sperm Conversely, other research, such as that by de Graaf et al (2007) and Alizadeh et al (2014), indicates that PUFA may not significantly affect semen quality across various species, including rams, stallions, and boars.
Conflicting results in previous studies highlight the impact of dietary fatty acids, particularly omega-3 and omega-6 polyunsaturated fatty acids (PUFAs), on sperm composition across various species The lipid and fatty acid profiles of sperm cells vary not only among different species but also among different animals, influencing the flexibility and compressibility of the sperm membrane This variation may affect the plasma membrane's ability to support the unique flagellar motion of sperm, ultimately impacting reproductive success.
Current research on the impact of a PUFA-enriched diet, rich in omega-6 and omega-3 fatty acids, on male buffalo reproduction, particularly in the contexts of India and Vietnam, remains limited Consequently, there is a pressing need for further studies to explore this important area.
Our hypothesis posited that supplementing male Murrah buffalo calves with omega-3 and omega-6 fatty acids would enhance hormone secretion and testis development, accelerate puberty onset, alter plasma membrane fatty acids, and improve spermatozoa and semen quality This study was conducted to achieve these specific objectives.
1 To evaluate the effect of dietary supplementation with omega-3 and omega-6 PUFA on the nutrient utilization, blood biochemical profile and the age of puberty in Murrah male buffaloes
2 To study the influence of omega-3 and omega-6 PUFA supplemented in diets on the quality of semen and spermatozoa
3 To assess the in vitro fertility of sperm obtained from Murrah buffalo bulls supplemented with omega-3 and omega-6 PUFA enriched diets
REVIEW OF LITERATURE
Male reproductive physiology
The male reproductive system comprises the testicles and secondary sex organs that facilitate the transport of spermatozoa to the female reproductive tract Key components include the epididymis, vas deferens, and penis, along with three accessory glands: the seminal vesicles, prostate, and Cowper’s gland A simplified diagram of this anatomy is presented in Figure 2.1.
Figure 2 1 The reproductive system of the bull (Ashwood, 2009)
The testes are paired, encapsulated ovoid organs in mammals that produce sperm cells and male hormones, including testosterone and dihydrotestosterone (Kenagy and Trombulak, 1986).
The seminiferous tubules, typically ranging from 200 to 350 µm in diameter, are enveloped by the tunica albuginea, a fibrous capsule rich in collagen and blood vessels (Polguj et al., 2011) The outer layer, known as the tunica vaginalis, is a mesothelium-lined sac that covers most of the testis except for its posterior border (Garriga et al., 2009) Fibrous septa extend from the tunica albuginea into the testis, creating lobules that house the convoluted seminiferous tubules In most mammals, these tubules open at both ends into the rete testis, a complex network of channels that facilitates the transport of spermatozoa and their surrounding fluid to the epididymis.
Before puberty, around 25 weeks of age, bull testes experience a significant growth spurt that continues until puberty, which occurs between 37 to 50 weeks This phase is marked by a notable increase in both the length and diameter of the seminiferous tubules, as well as the proliferation of germ cells into mature germ cells.
A significant increase in Leydig and Sertoli cells has been observed, indicating that average scrotal circumference is commonly utilized as a key indicator of puberty in bulls (Rawlings et al., 2008).
Research indicates that bulls with larger scrotal circumferences exhibit increased semen volume, higher sperm concentration, and a greater total sperm count per ejaculate (Latif et al., 2010) In practical terms, scrotal circumference serves as a reliable estimate of testicular size and sperm-producing tissue This measurement is taken using a flexible centimeter tape, wrapped snugly around the lowest part of the scrotum at its widest point, ensuring that the testes are fully descended to avoid inaccuracies caused by potential wrinkles in the scrotum.
The age of bulls significantly influences testicular development, particularly in young bulls aged 6 to 36 months During this critical growth period, especially between 6 and 16 months, there is rapid testicular growth, resulting in considerable variation in testis size among bulls of the same age within a breed This growth pattern is curvilinear rather than linear, with scrotal circumference typically increasing by 2 to 3 centimeters between one and two years of age across most breeds Research from Colorado State University indicates an adjustment factor of 0.026 centimeters per day, while Canadian researchers propose a specific formula for assessing scrotal growth.
Y1 = Actual SC when measures (centimeters)
X0 = This usually 365 (days), if adjusting to yearling age
X1 = Actual age in days when measured
Research shows a strong correlation (0.81) between scrotal circumference and sperm output in yearling bulls As scrotal circumference increases, there is a notable improvement in sperm motility, the percentage of normal sperm, sperm volume, concentration, and overall output, while the percentage of abnormalities decreases It is estimated that for every 1 centimeter increase in a sire's scrotal circumference, these positive effects on sperm quality are amplified.
Research indicates that male offspring can expect an increase of 0.25 in scrotal circumference (SC), as noted by Hahn et al (1969) A study on Murrah buffalo bulls by Pant et al (2003) found a strong correlation between SC and sperm output in younger animals, which diminishes with age This trend has also been observed in dairy bulls, supporting the findings of Hahn et al (1969).
Testicular biometric parameters are crucial for assessing domestic animals, especially those used in livestock production, as they play a vital role in andrological evaluations Scrotal circumference is one of the most frequently used metrics due to its ease of measurement and strong correlation with body weight Research indicates a significant relationship between scrotal circumference, age, and body weight (Pant et al., 2003; da Luz et al., 2013).
Table 2 1 Correlation coefficients between age, body weight and various testicular measurements in Murrah buffalo bull (Pant et al., 2003)
No Measurments Correlation between varous measurements
All correlation coefficients are significant (p < 0.01)
The epididymis, attached to the testicle, consists of three regions: the head, body, and tail The head and body play a crucial role in receiving sperm cells from the testicles and facilitating their maturation processes.
Sperm cells undergo maturation in the epididymis, taking approximately 5 to 11 days to traverse this region, as noted by Acott and Hoskins (1978) During this period, sperm cells are stored in the tail of the epididymis for up to 8 days, where they remain fertile until ejaculation or reabsorption occurs (Cornwall, 2009) The optimal conditions for sperm storage in the epididymal lumen include a rich supply of inorganic ions and organic molecules, an ideal testicular temperature, and reduced oxidative stress.
The vas deferens is a long, narrow tube that connects the epididymis to the pelvic urethra In animals, sterilization can be achieved by cutting a section of each ductus deferens, which prevents spermatozoa from being released during ejaculation This surgical procedure is known as vasectomy.
The male reproductive tract includes three accessory glands: the bulbourethral glands (Cowper’s glands), the prostate gland, and the seminal vesicles, which contribute 25%, 5%, and 50% of the total seminal plasma in bulls, respectively (Barszcz et al., 2011) These tubular branched lobular organs secrete fluids that form the majority of seminal plasma during ejaculation, providing a vital environment for spermatozoa Seminal plasma plays a crucial role in the survival of sperm cells within the female reproductive tract.
Semen is made up of spermatozoa and a liquid known as seminal plasma, which is primarily derived from the secretions of accessory glands The volume of semen and sperm count can vary significantly among bulls, with most typically ejaculating between 3 to 5 ml of semen that contains approximately 1 billion sperm per ml, totaling around 3 to 5 billion sperm per ejaculation After reaching sexual maturity, animals maintain continuous sperm production for the rest of their lives.
Characteristics of puberty and the role of testosterone hormone
During puberty, many species experience significant testicular growth, heightened gonadotrophin secretion, and increased testosterone production in response to LH surges, which triggers spermatogenesis The initiation of male reproductive capacity is marked by the release of the first spermatozoa, a process influenced by intricate interactions among the hypothalamus, anterior pituitary gland, and gonads Notably, the activation of interstitial (Leydig) cells occurs prior to this reproductive onset.
16 Review of literature formation of spermatozoa, with androgens conditioning the seminiferous tubules to gonadotropic stimulation (Figure 2.6)
Figure 2 6 Interrelationships between the testis, hypothalamus and pituitary gland in the control of male reproductive function
Puberty in bulls is marked by the production of at least 50 million sperm per ejaculate with a minimum of 10% linear motility, occurring at a scrotal circumference of approximately 27.9 cm The onset of puberty is influenced by factors such as breed, age, and environmental conditions, including food availability Buffalo generally achieve sexual maturity when they reach 55-60% of their final body weight The age at which buffalo reach puberty varies widely, with river buffalo typically maturing between 15 to 18 months and swamp buffalo between 21 to 24 months, depending on factors like nutrition and season.
17 Review of literature puberty at around 20-24 months of age (Chantaraprateep, 1987; McCool and Entwistle, 1989)
According to Ahmad et al (1989), the average age of puberty in Nili-Ravi buffalo bulls is 22.8 ± 1.1 months, with a body weight of 421 ± 19 kg and a testicular volume of 188 ± 12 cm³ Significant correlations (greater than 0.95, P < 0.01) exist among age, body weight, and testicular volume Notably, the growth in body weight and testicular size is significantly more pronounced between 8 to 15 months of age compared to other developmental stages Additionally, a cloned buffalo bull at NDRI, Karnal, achieved its first ejaculate at 18 months, weighing 450 kg, under intensive care and exceptional management.
830 g/day growth rate and 30 cm scrotal circumference (Dahiya and Pawan Singh,
Table 2 2 Puberty in water buffalo and related characteristics
Breed type Age of puberty (months)
River (Murrah) 32.40 408.60 24.50 Pant et al., 2003
River (Nili-Ravi) 22.80 421.00 23.80 Ahmad et al., 1989
River (Nili-Ravi) 24.00 450.00 26.00 Ahmad et al., 2010
River (Nili-Ravi) 26.50 515.00 26.30 Asghar et al., 1985
River (Nili-Ravi) 25.40 501.00 25.30 Heuer and Bajwa, 1986
Swamp (Australian) 30-33 >250.00 17-20 McCool and Entwistle, 1989 Swamp (Malaysian) 29.00 380.00 - Nordin et al., 1986
A study by Pant et al (2003) on Murrah buffalo bulls found a strong correlation between scrotal circumference (SC) and sperm output in younger animals, which diminished with age The authors suggest that to achieve optimal sperm output and potentially enhance fertility, Murrah bulls should have a mean SC of at least 24.4 cm.
27.3, 30.9, and 33.6 in the age groups of 18-24, 25-36, 37-48, 46-60 and > 60 months, respectively
The onset of puberty in buffalo bulls is regulated by the hypothalamus-pituitary-gonadal axis, primarily influenced by testosterone This hormone is released in an episodic manner, reflecting the pulsatile secretion of GnRH from the hypothalamus and LH from the anterior pituitary, playing a crucial role in spermatogenesis and the development of secondary sexual characteristics Research indicates that low testosterone levels are associated with delayed puberty in buffalo bulls, with values reported at 1.58 ± 0.32 ng/ml in young Murrah breed bulls Additionally, plasma testosterone concentration shows a positive correlation with HDL cholesterol levels Studies have shown that testosterone levels are low at birth (18.0 ± 2.9 pg/ml) and remain low until around 8 months of age, after which a significant increase occurs, marking the progression into puberty.
In buffalo bull calves, the pubertal period is observed to occur between 8 to 15 months of age, with a significant increase in serum testosterone levels noted at 8-9 months A second rise in testosterone concentrations occurs at 13-15 months, followed by a sharp increase, reaching an average of 376.2 ± 139.7 pg/ml by 17-19 months This steep rise in testosterone during 13-15 months may be attributed to increased testicular sensitivity to LH as the calves progress through puberty.
Overview of lipid and PUFAs
Lipids are a wide and diverse group of molecules that play a vital role in mammalian cell structure and function They are used as storage compounds, an
Lipids serve as vital energy sources and signaling molecules in mammalian cells, playing essential roles in membrane trafficking, protein regulation, and the formation of membrane sub-compartments (Shevchenko and Simons, 2010) They can be categorized into two main groups: simple and complex lipids Simple lipids yield one or two hydrolysis products, including tri-, di-, and mono-glycerides, free and esterified cholesterol, and free fatty acids (Noble, 1987; Christie, 1989) In contrast, complex lipids are primarily composed of phospholipids, which contain a phosphoric acid derivative that replaces a fatty acid in the molecule (Yeagle, 1987) The phosphate group can attach to various alcohols, resulting in crucial components of animal cell membranes, such as phosphatidyl inositol, phosphatidyl serine, phosphatidyl choline (lecithin), phosphatidyl ethanolamine, and sphingomyelin Overall, the predominant lipids in sperm cell membranes are phospholipids, sterols, and glycerides, with phospholipids being the most abundant.
Cell membrane fatty acids vary in composition based on carbon chain length, the number of double bonds, and the position of the first double bond from the methyl end Saturated fatty acids (SFA) lack double bonds in their hydrocarbon chain, whereas unsaturated fatty acids feature double bonds at different positions Each fatty acid comprises a hydrocarbon chain with a methyl group (CH3) at one end and a carboxyl group (COOH) at the other end.
Unsaturated fatty acids (UFA) are categorized into monounsaturated fatty acids (MUFA), which have a single double bond, and polyunsaturated fatty acids (PUFA), which contain multiple double bonds Additionally, MUFA and PUFA are divided into three families—omega-3, omega-6, and omega-9—based on the positioning of the first double bond relative to the methyl terminal.
n-3 polyunsaturated fatty acids (PUFAs) are characterized by their first double bond located on the third carbon from the methyl end of the fatty acid chain In contrast, n-6 PUFAs have their first double bond on the sixth carbon, while n-9 fatty acids feature a double bond on the ninth carbon Key n-3 PUFAs found in sperm include Alpha-Linolenic acid (ALA), Docosahexaenoic acid (DHA), Docosapentaenoic acid (DPA), and Eicosapentaenoic acid (EPA) Notable n-6 PUFAs present in sperm cells are Arachidonic acid (AA) and Linoleic acid (LA), with Oleic acid (OA) being the primary n-9 fatty acid.
N-3 and n-6 fatty acids are essential components of plant lipids, with n-6 fatty acids primarily sourced from dietary plant oils like palm, sunflower, corn, and soybean oils In contrast, n-3 fatty acids are abundant in green leafy vegetables, seeds such as flaxseed and linseed oil, nuts, and legumes, while small amounts can also be found in corn, sunflower, and safflower oils (Kromhout et al., 2012).
Early research on ruminal lipid metabolism focused on the fate of fatty acids as they traversed the rumen and interacted with the microbial population Findings revealed two primary processes occurring in the rumen: the hydrolysis of ester linkages in lipids and the biohydrogenation of unsaturated fatty acids.
In ruminants, the metabolism of dietary lipids begins in the rumen with the hydrolysis of ester linkages in triglycerides, phospholipids, and glycolipids This process is facilitated by enzymes such as lipases, galactosidases, and phospholipases produced by bacteria and protozoa, leading to the production of free fatty acids and glycerol, while mono- and di-glycerides accumulate minimally Additionally, glycerol is fermented quickly within the rumen.
Research indicates that propionic acid is a significant end product of fermentation (Garton et al., 1961) While hydrolysis typically exceeds 85%, various factors can influence its rate and extent For instance, higher dietary fat levels can decrease hydrolysis efficiency (Beam et al., 2000), and conditions such as low rumen pH and the presence of ionophores can inhibit bacterial growth and activity (Van Nevel and Demeyer, 1995; Demeyer and Doreau, 1999).
The rumen hosts two key processes: the hydrolysis of ester linkages in lipids and the biohydrogenation of unsaturated fatty acids This involves the breakdown of triglycerides (TG) and glycolipids (GL) into fatty acids (FA), as outlined by Davis (1990).
Biohydrogenation of unsaturated fatty acids is a key process in the rumen, occurring after lipolysis has released free fatty acids This transformation is influenced by factors affecting hydrolysis, as unsaturated fatty acids have short half-lives in the rumen due to rapid microbial hydrogenation into more saturated forms The initial step in this process often involves the isomerization of the cis-12 double bond to a trans-11 configuration.
22 Review of literature configuration resulting in a conjugated di- or trienoic fatty acid (Harfoot and Hazlewood, 1997) Next is a reduction of the cis-9 double bond resulting in a trans-
The hydrogenation process of fatty acids culminates in the formation of stearic acid (C18:1 trans-15) from the trans-11 double bond However, in the presence of high levels of n-6 polyunsaturated fatty acids (PUFAs), this hydrogenation often halts before reaching stearic acid, resulting in a variety of cis and trans isomers of monoenoic fatty acids.
1973) The most important is trans-vaccenic acid (C18:1)
Most biohydrogenation, exceeding 80%, occurs with fine food particles due to the extracellular enzymes of bacteria, either associated with feed or free in suspension The rumen biohydrogenation rate of fatty acids increases with unsaturation, with linoleic (n-6 PUFA) and linolenic acids (n-3 PUFA) being the primary substrates Typically, n-6 PUFA and n-3 PUFA are hydrogenated by 70-95% and 85-100%, respectively However, this extensive hydrogenation decreases with high-concentrate diets due to inhibited lipolysis at low pH levels Additionally, the presence of excessive unprotected fatty acids negatively impacts hydrogenation, highlighting the need for effective strategies to protect these essential fatty acids from ruminal degradation.
After leaving the rumen, dietary fat travels to the small intestine, where pancreatic lipase breaks down triacylglycerols into primarily 2-monoacylglycerols and free fatty acids These components are then absorbed with the help of the lipoprotein lipase enzyme following micelle formation, allowing for widespread distribution throughout the body’s tissues.
Unlike non-ruminants, ruminants do not absorb dietary fatty acids intact due to biohydrogenation by rumen microbes, making the use of rumen-protected fat essential to safeguard polyunsaturated fatty acids from microbial degradation Once absorbed into cell tissues, Alpha-Linolenic acid (C18:3 n-3) undergoes conversion processes that are crucial for maintaining the health and function of ruminant animals.
The literature review highlights the significance of important fatty acids, including DHA (C22:6n-3), DPA (C22:5n-3), EPA (C20:5n-3), and linolenic acid (C18:2n-6), which are converted into arachidonic acid (C20:4n-6), docosatetraenoic acid (C22:3n-6), and docosapentaenoic acid (C22:5n-6) through a series of elongation and desaturation reactions (Lenzi et al., 1996) These fatty acids play crucial roles in various local tissues, contributing to metabolism, growth, development, reproduction, and overall production in animals.
Figure 2 8 Lipid metabolisms in ruminants (Tanaka, 2005)
Pa rtial ly b y p assin g b iohy drog ena tion
TISSUES C18:0 C18:3 Trans -11 C18:1 Cis-9, tran-11 C18:2 ∆9 desaturase
Pa rtial ly byp a ssing biohydrog ena tion
2.3.3 Effect of rumen protected fat (bypass fat) on digestibility and utilization of dietary nutrients by ruminants
Oxidative stress and its relationship with PUFA and sperm quality
All cells require oxygen (O2) for efficient energy production in mitochondria, where over 85% of cellular oxygen is utilized by the mitochondrial electron transport system However, this process also generates reactive oxygen species (ROS), as 1-3% of electrons escape the transport chain, leading to the formation of superoxide Under normal physiological conditions, molecular oxygen undergoes reactions that produce superoxide anion (O2-), hydrogen peroxide (H2O2), and water (H2O) Oxygen acts as the primary oxidant in metabolic reactions that extract energy from various organic molecules Consequently, oxidative stress arises from these oxygen-dependent metabolic processes, representing an imbalance between prooxidant and antioxidant systems.
Oxidative stress is defined as a condition where intact prooxidant and antioxidant systems in cells are overwhelmed, leading to an imbalance during normal aerobic metabolism (Sies, 1991; Halliwell, 2006) This imbalance can result from increased free radical generation or decreased antioxidant levels, which can cause oxidative damage to lipids, proteins, carbohydrates, and nucleic acids, ultimately resulting in cell death under severe oxidative stress (Guyton and Kensler, 1993).
Antioxidants are substances that prevent or repair oxidative damage to molecules, as defined by Halliwell and Gutteridge (2007) Under normal physiological conditions, free radicals and reactive oxygen species (ROS) are regulated by various cellular defense mechanisms, which include both enzymatic antioxidants (such as Catalase, Glutathione Peroxidase, and Superoxide Dismutase) and non-enzymatic antioxidants (like vitamins E and C, and glutathione) (Lopaczynski and Zeisel, 2001) Enzymatic antioxidants, also referred to as natural antioxidants, play a crucial role in neutralizing excess ROS, thereby protecting cellular structures from damage Consequently, these enzymes are essential for assessing the antioxidant activity within cells and tissues.
Superoxide Dismutase (SOD): SOD was first isolated by Mann and Keilin
In 1938, a protein believed to store copper was identified and later named erythrocuprein, indophenol oxidase, and tetrazolium oxidase However, its catalytic function was uncovered by McCord and Fridovich in 1969, revealing that superoxide dismutase (SOD) catalyzes the conversion of superoxide into hydrogen peroxide and oxygen.
Catalase (CAT) is a heme-containing enzyme that plays a crucial role in breaking down hydrogen peroxide into water and oxygen Located in peroxisomes, catalase manages the cytosolic and mitochondrial peroxides produced during urate oxidation Additionally, mitochondrial superoxide dismutase (SOD) efficiently transforms most mitochondrial superoxide ions into hydrogen peroxide, working in tandem with catalase to safeguard cells from damage caused by highly reactive hydroxyl radicals.
A review of literature indicates that hydrogen peroxide (H2O2) production increases with elevated superoxide dismutase (SOD) activity during heat stress (Miyazaki et al 1991; Bernabucci et al 2002) This rise in H2O2 is associated with a coordinated increase in plasma concentrations of catalase and glutathione peroxidase, highlighting a significant positive correlation between catalase and SOD activities.
Glutathione peroxidase (GPx) is a selenium-dependent antioxidant enzyme that plays a crucial role in converting hydrogen peroxide (H2O2) into water During heat stress, the heightened activity of superoxide dismutase (SOD) leads to an increased production of H2O2, which in turn triggers a coordinated rise in GPx levels to mitigate oxidative stress.
Research on the impact of bypass fat on antioxidant enzyme activity related to oxidative stress in growing buffalo and fertility is lacking Most studies on ruminants suggest that stress conditions, whether from thermal stress or the physiological state of the animal, lead to an increased production of superoxide radicals.
2.4.3 Relationship between lipid peroxidation and PUFA
Lipid peroxidation is a process where oxidants, including free radicals, attack lipids with carbon-carbon double bonds, particularly polyunsaturated fatty acids (PUFAs), leading to the formation of lipid peroxyl radicals and hydro peroxides The occurrence of lipid peroxidation is influenced by the levels of antioxidant enzymes and the fatty acid composition in organisms, as well as physiological changes related to development, production, age, and sex.
The overall process of lipid peroxidation consists of three steps: initiation, propagation, and termination (Yin et al., 2011; Girotti, 1998; Kanner et al., 1987)
Lipid peroxidation begins with prooxidants, such as hydroxyl radicals, which remove allylic hydrogen to create carbon-centered lipid radicals (L*) During the propagation phase, these lipid radicals swiftly react with oxygen, leading to the formation of lipid peroxides.
The peroxy radical (LOO*) initiates lipid peroxidation by abstracting hydrogen from lipid molecules, resulting in the formation of a new lipid radical (L·) and lipid hydroperoxide (LOOH) This chain reaction continues until antioxidants, such as vitamin E, intervene by donating a hydrogen atom to the LOO* species, creating a vitamin E radical that reacts with another LOO*, ultimately leading to the formation of non-radical products Once lipid peroxidation begins, a series of chain reactions ensues until termination products are generated.
Lipid peroxidation occurs when oxygen reacts with unsaturated lipids, resulting in a diverse range of oxidation products The primary products of this reaction are lipid hydroperoxides (LOOH), while secondary products include malondialdehyde (MDA), propanal, hexanal, and 4-hydroxynonenal.
MDA, a byproduct of Arachidonic acid and larger polyunsaturated fatty acid (PUFA) decomposition, is formed through both enzymatic and non-enzymatic processes It has long been recognized as a reliable biomarker for assessing lipid peroxidation in omega-3 and omega-6 fatty acids due to its ease of measurement.
Lipid peroxidation is often assessed through the measurement of Thiobarbituric Acid Reactive Substances (TBARS), which serve as a marker of oxidative stress in plasma (Esterbauer et al., 1990; Pryor, 1989) While the Thiobarbituric acid test is a reliable indicator of oxidative stress, it is not exclusively a marker of lipid peroxidation (Halliwel and Chirico, 1993) The oxidation of unsaturated fatty acids by reactive oxygen species (ROS) in the presence of iron produces lipid-centered radicals via the Fenton reaction, leading to the formation of lipid hydro-peroxides (Trevisan et al., 2001) This process results in reduced membrane fluidity and increased fragility of erythrocyte membranes (Chen and Yu, 1994) Notably, TBARS concentrations in erythrocytes have been found to rise in cattle and buffalo exposed to heat (Ashok et al., 2007).
High levels of dietary polyunsaturated fatty acids (PUFAs) can increase cell vulnerability to oxidative damage from free radicals, highlighting the essential role of antioxidants in protecting PUFAs and cell membrane components from oxidation Spermatozoa are particularly unique due to their structure and function, making them especially susceptible to lipid peroxidation (LPO) damage Unlike other cells, spermatozoa cannot repair damage caused by excessive reactive oxygen species (ROS) because they lack the necessary cytoplasmic enzyme systems, further emphasizing their heightened sensitivity to oxidative stress.
Effect of PUFA on production of hormone and testis development
Fatty acids play a crucial role in the phospholipid layer of cell membranes, influencing membrane properties and component interactions (Rooke et al., 2001) In males, the reproductive system is regulated by the hypothalamic-pituitary-testicular axis, which governs hormones essential for testicular development and spermatogenesis Research indicates that polyunsaturated fatty acids (PUFAs) and their derived eicosanoids have a direct relationship with this hormonal axis (Jump and Clarke, 1999; Ojeda et al., 1981; Free et al., 1980; Saksena et al., 1978) Therefore, investigating the impact of dietary PUFAs on the secretion of GnRH, LH, and FSH, as well as the hormonal responsiveness of these cells, is of significant importance (Surai et al., 2000).
Esmaeil et al (2012) studied the impact of various dietary fatty acids on semen quality and blood parameters in rams supplemented with 35 g per day of Palm oil, Sunflower oil, and fish oil The results indicated that rams receiving fish oil exhibited the highest testosterone concentration in the blood at 11.3 ng/ml, compared to 10.8 ng/ml for Sunflower oil and 10.2 ng/ml for Palm oil This suggests that fish oil, rich in omega-3 polyunsaturated fatty acids (PUFAs), may enhance the phospholipid composition of testicular plasma membranes, thereby affecting gonadotropin receptor expression and testosterone synthesis Conversely, the study also noted the effects of dietary Soybean oil and fish oil at a 2% dry matter level.
A study by Adibmoradi et al (2012) found that control treatments did not significantly impact testosterone levels in male growing kids However, a diet supplemented with fish oil notably enhanced testicular growth metrics—including circumference, volume, width, and length—as well as increased the counts of seminiferous tubules, Leydig cells, Sertoli cells, spermatogonia, and spermatocytes, along with absolute fresh testis mass at slaughter, compared to control or soybean oil diets This suggests that fish oil, rich in n-3 polyunsaturated fatty acids (PUFAs), positively influences gonadal development, with higher dietary DHA levels correlating to improved testis development The beneficial effects of n-3 PUFAs from fish oil on testis development may be linked to their influence on the hypothalamo-pituitary-gonadal axis, which is crucial for testis development in males through the actions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) FSH is vital for Sertoli cell proliferation, while LH plays a key role in the differentiation and maturation of Leydig cells.
Research indicates that GnRH treatment accelerates puberty onset and boosts Sertoli cell counts, sperm production, and testicular weight (Chandolia et al., 1997) Additionally, Bagu et al (2004) found that administering bFSH to bull calves promotes testicular growth, speeds up puberty onset, and improves spermatogenesis.
It has been suggested that IGF-I is one of the important factors for germ cell development, maturation and the motility of the spermatozoa (Hoeflich et al., 1999)
A recent study by Selvaraju et al (2009) found that the addition of IGF-I in vitro significantly enhances the quality of buffalo spermatozoa by improving sperm viability and motility, primarily through its antioxidant properties and by maintaining membrane integrity Moreover, polyunsaturated fatty acids (PUFAs) are linked to IGF-I's role in regulating testicular development, as highlighted by Fair et al (2014), which underscores the importance of these compounds in reproductive health.
In a study examining the effects of PUFA fish oil supplementation, rams receiving this treatment exhibited significantly higher IGF-I concentrations by week 9, following 64 days of feeding, compared to the control group that was given saturated palmitic acid This outcome aligns with the findings of Robinson et al (2002), reinforcing the positive impact of PUFA on IGF-I levels.
The literature review highlights the increased levels of IGF-I in bovine body fluids, focusing on whether reproductive tissues produce IGF-I and the influence of PUFAs on its production Local IGF-I production is crucial for tissue growth, impacting the proliferation of precursor Leydig cells, the establishment of a normal population of adult Leydig cells, and effective steroidogenesis The testis is known to secrete IGF-I, and it has been found in bovine seminal plasma, with IGF-I receptors expressed in spermatogonia, spermatocytes, spermatids, and spermatozoa.
So far, the mechanism that PUFAs may affect the IGF-I concentration in testis tissue has been not elucidated yet.
Effect of PUFAs on spermatozoa and semen quality
Gulliver et al (2012) highlighted the need for direct feeding studies on n-3 polyunsaturated fatty acids (PUFA) to assess their impact on male fertility in ruminants Research indicates that PUFA are crucial components of sperm cell membranes, influencing sperm quality through their physiological properties Incorporating PUFA into the diet alters the fatty acid profile of sperm plasma membranes, leading to enhanced sperm quality Kelso et al (1997) emphasized that PUFA significantly affect sperm lipid metabolism, motility, and fusion with oocytes A decline in n-3 PUFA levels in sperm lipids correlates with reduced sperm count and motility in older bulls Studies involving Holstein bulls (Gholami et al., 2010), goat bucks (Dolatpanah et al., 2008), and rams (Samadian et al., 2010) further demonstrated that including n-3 sources in their daily diet improved sperm quality, both fresh and post-cryopreservation.
A study by Adeel et al (2009) investigated the effects of three fat sources on Nili-Ravi buffalo bulls over a 63-day period The bulls were fed a balanced ration, a sunflower oil-supplemented ration (SFO), and a whole sunflower seeds-supplemented ration (SFS).
Table 2 5Effect of dietary PUFA enriched fat sources on semen quality
Reference Species Fat source Percent inclusion
Research indicates that the addition of various oils to ram diets can influence sperm characteristics Effect de Graaf et al (2007) found that incorporating 5% sunflower oil had no impact on the motility, viability, or acrosome integrity of sex-sorted sperm In contrast, Samadian et al (2010) reported that a 3% inclusion of fish oil significantly increased the proportion of DHA in sperm fatty acid composition, leading to improvements in sperm concentration and motility Additionally, Jafaroghli et al (2014) explored the effects of fish oil on ram sperm, further contributing to the understanding of dietary influences on reproductive health.
Improved seminal quality, increased motility, HOST and percentage of sperm with normal acrosome
Esmaeili et al (2012) Ram Palm oil, sunflower oil, fish oil
35g/day Improved refreezing semen characteristics after thawing, 35 days after the removal of fatty acid source, the percentage of C22:6 was highest in the fish oil treated group
Alizadeh et al (2014) Ram Fish oil &
Dietary FO had significant positive effects on all sperm quality and quantity parameters compared with the control during the feeding period
Dolatpanah et al (2008) Goat Fish Oil
Improved testes development Enhanced the quality and quantity of goat semen
Fair et al (2014) Ram Protected fish oil
2% Increased semen concentration but no effect on other semen quality parameters including semen volume, wave motion, and progressive linear motion
Adeel et al (2009) Buffalo Sunflower oil,
1% Improved the quality of sperm including motility and HOS of post-thawed sperm Moallem et al (2015) Bull Flaxseed oil
Changed in the characteristics of both fresh and frozen- thawed semen, increased motility and progressive motility of sperm
Inclusion of sunflower oil or whole sunflower seeds at up to 1% of dry matter intake (approximately 0.13 kg of fat per day) is safe for breeding buffalo bulls and does not negatively impact the quality of fresh and frozen-thawed semen Bulls that were fed sunflower-enriched diets exhibited improved motility and plasma membrane integrity, as measured by hypo-osmotic swelling (HOS), in their post-thawed spermatozoa compared to those on a control diet These findings suggest that sunflower oil or whole sunflower seeds can mitigate the detrimental effects of cryopreservation on sperm plasma membranes, as supported by research from Khan and Ijaz.
In 2007, it was reported that damage to the sperm plasma membrane during cryopreservation significantly contributes to the reduced motility of frozen-thawed spermatozoa, a finding that aligns with the research conducted by Milovanov and Golubj.
(1973), who reported feeding of Soya bean oil, high in Linoleic acid, improved post-thaw motility in rams
Calisici (2010) found that dietary supplementation with 800 g/day of Alpha-Linolenic acid (ALA) and 400 g/day of Palmitic acid (PA) improved sperm quality in Holstein Friesian bulls, increasing DHA levels in ALA-supplemented bulls while PA bulls showed no change Moallem et al (2015) further examined the effects of different omega-3 fatty acids on bull sperm, supplementing diets with 360 g/day of saturated fatty acids, 450 g/day of Flaxseed oil (providing 84.2 g/day of C18:3n-3), and 450 g/day of fish oil (providing 8.7 g/day of C20:5n-3 and 6.5 g/day of C22:6n-3) Their results indicated that the incorporation of n-3 fatty acids into sperm began after six weeks of supplementation, with Flaxseed oil showing superior motility and progressive motility in frozen-thawed semen compared to fish oil and saturated fatty acid groups (55.5% versus 50.9%, 47.7%, and 43.4% versus 38%, 37%, respectively) Notably, despite the higher DHA content in fish oil, sperm quality was lower than that of the Flaxseed oil group.
DHA, being highly sensitive to oxidation, may exceed optimal levels in the sperm of fish oil (FO) bulls, hindering improvements in sperm quality compared to those from the Flaxseed oil group The n-6 to n-3 PUFA ratio was significantly lower in bulls supplemented with fish oil, measuring 0.5% versus 1.01% for saturated fatty acids and 0.66% for Flaxseed oil, aligning with findings by Calisici (2010) Dietary variations in PUFA, particularly DHA, significantly alter sperm fatty acid composition, as shown in table 2.6 Incorporating DHA into sperm enhances survival, motility, and progressive motility of frozen-thawed sperm, emphasizing the importance of feeding dietary n-3 PUFA during early spermatogenesis for effective incorporation into sperm lipids (Samadian et al., 2010).
Heat stress negatively impacts sperm quality in Holstein bulls, but dietary omega-3 supplementation can enhance the in vitro quality and motility of fresh semen, although its effects are less pronounced in frozen-thawed semen (Hamid et al., 2010) Polyunsaturated fatty acids (PUFAs) may increase oxidative stress, potentially compromising semen quality (Wathes et al., 2007) In goats, dietary fish oil (2.5% DM) and vitamin E (0.3 g/kg DM) supplementation improved both the quality and quantity of semen, highlighting the role of vitamin E in preventing peroxidation of polyunsaturated fatty acids and reducing free radical production (Dolatpanah et al., 2008; Strzezek et al., 2004) Similar findings were reported in rams, where fish oil and vitamin C supplementation also yielded positive effects on semen quality (Alizadeh et al., 2014; Jafaroghli et al., 2014).
Table 2 6 Effect of PUFAs on alteration of fatty acid composition of sperm lipid
Reference Species Item PUFAs (g/100g FA)
FO Fresh sperm -1 st Week -13 th Week
FO Fresh sperm -1 st Week -12 th Week
CO (control diet), FO (fish oil) and FLX (flaxseed oil) *P < 0.05; **P < 0.01 a,b,c Within columns, means with different letter superscripts are statistically different (P < 0.05)
DM had a significant increase of the proportion of DHA in spermatozoa, motility, HOS and percentage of sperm with normal acrosome, which may have beneficial effects on fertility
Fair et al (2014) found that supplementing rams' diets with 2% fish oil effectively altered the plasma n-6/n-3 ratio but had minimal impact on sperm fatty acid profiles While this supplementation increased semen concentration, it did not enhance the quality of liquid-stored semen Similarly, de Graaf et al (2007) reported that rams receiving 5% sunflower oil, rich in linoleic acid, showed no improvement in the cryosurvival of ram spermatozoa.
In addition to the sources and types of PUFA supplements, effect of PUFA supplementation in diet also depends upon the quantity and ratio of n-6/n-3 PUFA
Research indicates that both humans and livestock evolved on a diet with an n-6 to n-3 PUFA ratio of 1:1, but modern dietary habits have skewed this ratio significantly, particularly in western populations, where it now ranges from 10:1 to 25:1 (Simopoulos, 1991) While numerous studies have explored the impact of PUFAs on reproductive performance in ruminants, such as rams (Fair et al., 2014), buffalo (Adeel et al., 2009), and bovines (Moallem et al., 2015), there is limited information on the effects of optimal n-6/n-3 ratios specifically in male ruminants Am-in et al (2011) found that lower n-6/n-3 ratios in boar sperm correlated negatively with sperm motility, viability, and morphology, highlighting the significance of the right PUFA balance for sperm quality Liu et al (2015) identified an optimal ratio of 6.6 in boars with vitamin E supplementation and 1 without it (Lin et al., 2016) Additionally, Yan et al (2013) emphasized the importance of a balanced n-6/n-3 ratio in male rat reproduction, establishing an optimum ratio of 0.66 compared to other tested ratios Despite the limited data available, these findings underscore the necessity of maintaining appropriate n-6/n-3 PUFA ratios for male reproductive health.
Recent literature on male ruminant reproduction highlights the significance of the n-6/n-3 PUFA ratio in improving sperm quality Studies indicate that an optimal ratio of 0.96 enhances ram sperm quality (Samadian et al., 2010; Jafaroghli et al., 2014), while bull sperm shows better quality at ratios of 2.01 (Moallem et al., 2015) and 2.39 (Khoshvaght et al., 2015) Notably, Moallem et al (2015) found that a ratio of 2.01 positively influenced sperm quality more than a higher ratio of 3.96 This underscores the need for further research to better understand the implications of PUFA ratios in male ruminants.
Recent feeding experiments have aimed to alter the fatty acid composition of sperm membranes through PUFA supplementation to enhance sperm quality and fertility across various livestock species Studies indicate that dietary modifications can effectively change the fatty acid profile of sperm membranes, leading to improved sperm quality; however, results have been inconsistent This variation may stem from differences in the type and amount of dietary fats, particularly omega-3 and omega-6 PUFA, as well as species-specific differences in PUFA content within semen and spermatozoa For instance, bovine semen contains higher levels of DHA and glutathione peroxidase compared to equine and porcine semen, which may explain the differing impacts of PUFA supplementation on sperm quality Additionally, there is a lack of comprehensive studies on buffalo bulls, particularly in India, highlighting the need for further research in this area.
The materials used and experimental techniques during the study are presented as below in this chapter
3.1.1 Selection and distribution of animals
Eighteen male buffalo calves, averaging 9.04 ± 0.07 months in age and 146.06 ± 0.61 kg in body weight, were selected from the Cattle Yard of the National Dairy Research Institute in Karnal They were randomly divided into three groups of six each: control, PFA, CaSFA, and CaLFA The experiment was conducted with the approval of the Animal Ethics Committee at NDRI, India Detailed information about the animals at the start of the experimental feeding is provided in Table 3.1.
Table 3 1Distribution of animals in different treatment groups
Groups Animals/group Body weight (kg) Age (month)
3.1.2 Housing and management of animals
The male buffalo calves were kept in well-ventilated pens with individual feeding facilities, ensuring cleanliness for both the animals and their sheds throughout the experiment Daily exercise was facilitated by allowing the calves to roam freely for half an hour each morning A deworming schedule was implemented at the start of the experiment and continued every six months thereafter.
After a three-week adaptation period, the animals were transitioned to their designated experimental diets, adhering to the ICAR (2013) feeding standards to fulfill their nutritional needs The diet consisted of a concentrate mixture and green fodder, adjusted based on the availability of fodder and wheat straw Daily records were meticulously kept of the feed provided and any leftovers.
Table 3 2 Composition of diet (on DM basis)
Calsium salt of Soybean oil 4.67
Calcium salt of Linseed oil 4.67