Seminal Proteins Hint at Sexual Maturity and Feed Efficiency in Young Beef Bulls

Background

The proteome is the entire set of proteins produce by a cell, tissue, or complete organism. Proteomics is the study of the structure, function and interactions of proteins in a diversity of biological samples. There is a growing interest in the proteins associated with fertility (FAP) that are present in seminal plasma of bulls. These proteins are essential for sperm survival, sperm fertilizing capacity and other functions (Table 1). These proteins are also related to puberty onset and sexual maturity; in fact, there are studies proposing the use of FAP as indicators of bull fertility (Kumar et al., 2012).

A recent publication from our group provides evidence of an antagonistic relationship between improved feed efficiency and measures related to fertility in young bulls (Fontoura et al., 2016). The "repro-traits" evaluated were semen quality, testis texture (via ultrasound), testis micro-structure (via histology) and scrotal circumference. We also collected seminal plasma of the same bulls, and using proteomics, furthered our understanding of the associations between the following:

  1. fertility-related measures with seminal proteins
  2. feed efficiency and seminal plasma proteins

Table 1. Selected fertility associated proteins (FAP) and respective metabolic functions.

Protein
Metabolic functions
Acidic seminal fluid protein (aSFP) Associated with sperm motility, metabolism & membrane integrity
Acrosin (ACR) Positively associated with sperm motility and semen concentration
Cathepsin D (CathD) Promotes seminiferous tubule maturation
Epididymal sperm-binding (ESBP1) Binds and tags sperm cells which would be dead upon ejaculation
Glutathione peroxidase (GPX) Promotes homeostasis and prevents cellular oxidative stress
Niemann-Pick type C2 (NPC2) Related to sperm cholesterol metabolism and semen concentration
Osteopontin (OPN) Facilitates the acrosome reaction and sperm-oocyte interaction
Phosphoglycerate kinase 2 (PK2) Promotes homeostasis and sperm motility, prevents oxidative stress
Prosaposin (PSAP) Associated with the structural development of the testis
Protein C inhibitor (PCI) Protects sperm from premature acrosome reaction and degradation
Seminal plasma protein 1 (BSP1) Similar to BSP3, also regulates sperm energy use for motility
Seminal plasma protein 3 (BSP3) Binds sperm, forms sperm reservoir in the female reproductive tract
Spermadhesin Z13 (SZ13) Has sperm decapacitating properties, related to low fertility

Bull evaluation, sampling and proteomic analysis

Crossbred bull calves sourced from both the Elora Beef Research Center and New Liskeard Research Station were fed a corn-based ration and submitted to a routine performance evaluation with assessment of individual feed intake. Feed efficiency was determined using residual feed intake (RFI) adjusted for body composition, and bulls were grouped as efficient (low-RFI) or inefficient (high-RFI).

At an average age of 12.5 months bulls were submitted to a breeding soundness evaluation, along with testis ultrasonography. A portion of the semen collected was prepared for proteomic evaluation. Analyses were conducted at the SPARC BioCentre (Hospital for Sick Children, Toronto). Over 500 proteins were identified in seminal plasma of the bulls and a portion of them were evaluated in more detail based on the existing scientific literature on fertility associated proteins (FAP).

At slaughter, the reproductive tract was collected and dissected to obtain testis and epididymis weight. Testis samples were also collected for histological analysis, which determined the abundance of seminiferous tubules at distinct stages of maturity, see Figure 1.

What are the seminal proteins telling us?

Seminal proteins are associated with several biological processes and reproductive tissue development. Table 1 summarizes some of these aspects relevant to young bulls. The seminal proteins PK2, PSAP and SZ13 are associated with reproductive organ development. Our study showed significant correlations between some of these proteins and scrotum circumference, testis weight and epididymis weight (Table 2). Interestingly, PK2 seems to play a role in reproductive organ development and size in addition to its known functions in cell homeostasis and motility scores. In the case of PSAP, the opposite association with organ size might be explained by its activity in the proper development of reproductive organs. Basically, smaller reproductive organs still in development are associated with higher amounts of PSAP. The negative associations demonstrated between SZ13 and fertility-related measures are expected, as this is an anti-fertility protein and organ size is highly related to sperm-producing capacity.

Table 2. Correlations between seminal proteins and reproductive organ development (%)

Traits
PK2
PSAP
SZ13
Scrotal circumference
56
-38
-66
Testis weight
58
-40
-63
Epididymis weight
39
-48
-65

*Correlations can range from -100% to +100%. Negative and positive values denotes antagonism and synergism, respectively.

Sperm motility was positively correlated with both aSFP (39%) and BSP1 (45%). These proteins are related to energy use and conservation by the sperm and relate to the fertilizing capacity of the sperm. Semen concentration was positively associated with ACR (49%) and NPC2 (37%). These proteins have a well-known positive relationship with semen concentration in other species as well. Thus, seminal plasma with higher aSFP, BSP1, ACR and NPC2 is associated with better semen quality in bulls.

Testis structure was assessed using testis ultrasonography and is positively associated with BSP1 (43%) and SZ13 (40%). These associations are probably due to the fact that these abundant proteins in the seminal plasma can increase the echogenicity of the organ, making the ultrasound images whiter. These observations are of practical relevance, since testis ultrasonography is a quick and easy assessment and can be conducted on commercial farms.

Testis microstructure (histology analysis of seminiferous tubules that house the sperm production) was correlated with several proteins (Figure 1). This is important, as testis microstructure is an indicator of sexual maturity. The association between CathD and seminiferous tubule maturity is demonstrating that this protein, along with BSP3, may aid in proper development of testis microstructure. In the same way, OPN and GPX are associated with mature tubules. Previous studies indicate that OPN is associated with better sperm performance, while GPX helps overcome oxidative stress that is detrimental to semen quality.

Correlations (%) between abundance of proteins and maturity of seminiferous tubules found in testis of young bulls. Images are cross-sections of seminiferous tubules, 400x magnification.

Figure 1. Correlations (%) between abundance of proteins and maturity of seminiferous tubules found in testis of young bulls. Images are cross-sections of seminiferous tubules, 400x magnification.

Text Equivalent of Figure 1

In addition to the associations between the structure of reproductive organs and semen quality, seminal proteins are also associated with feed efficiency. Figure 2 shows the relative abundance of seminal proteins in efficient and inefficient bulls. Three of these proteins (ESBP1, GPX and NPC2) are associated with diminished semen quality. ESBP1 is known to be strongly associated with the number of dead sperm in the ejaculate and NPC2 is a precursor of this protein. There is evidence indicating that increased GPX is also associated with certain sperm defects. Conversely, PCI is associated with sperm preservation and improved fertility. These results indicate that inefficient bulls had a more desirable profile of seminal proteins related to fertility, compared to the efficient bulls.

Different protein expression among young bulls by feed efficiency groupings

Figure 2. Different protein expression among young bulls by feed efficiency groupings

Text Equivalent of Figure 2

Take home messages

  • Fertility-associated proteins from seminal plasma of young bulls relate to sexual maturity based on fertility-related traits (organ size, sperm quality and testis ultrasound and histology).
  • Key fertility-associated proteins differ between feed efficient and inefficient young bulls. These proteins should be considered in the evaluation of feed efficiency and fertility potential.
  • The differences in seminal proteins between feed efficient and inefficient young bulls supports the association of delayed sexual maturity and improved feed efficiency.
  • Research should focus on strategies to identify bulls with high feed efficiency accompanied by early sexual maturity and proven fertility still as young bulls.

Acknowledgements

We thank the following organizations for financial support: Beef Cattle Research Council, Agriculture and Agri-Food Canada, Beef Farmers of Ontario, Canadian Simmental Association and Ontario Ministry of Agriculture, Food and Rural Affairs. The assistance from the staff at the Elora Beef Research Centre is also acknowledged.

References

Fontoura ABP, Montanholi YR, Diel de Amorim M, Foster RA, Chenier T, Miller SP. Associations between feed efficiency, sexual maturity and fertility related measures in young beef bulls. Animal 2015; 10:95-106.

Kumar P, Singh I, Yadac PS. Seminal plasma proteome: promising biomarkers for bull fertility. Agricultural Research 2012; 1:78-86.


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